A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center

The ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. Here we demonstrate that the ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg²⁺-μc's). Common features of Mg²⁺-μc's include two paired Mg²⁺ ions that are chelated by a common bridging phosphate group in the form Mg_(a)²⁺–(O1P-P-O2P)–Mg_(b)²⁺. This bridging phosphate is part of a 10-membered chelation ring in the form Mg_(a)²⁺–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg_(a)²⁺. The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg²⁺ ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups. The Mg²⁺-μc's in the LSU (large subunit) appear to be highly conserved over evolution, since they are unchanged in bacteria (Thermus thermophilus, PDB entry 2J01) and archaea (Haloarcula marismortui, PDB entry 1JJ2). The 2D elements of the 23S rRNA that are linked by Mg²⁺-μc's are conserved between the rRNAs of bacteria, archaea and eukarya and in mitochondrial rRNA, and in a proposed minimal 23S-rRNA. We observe Mg²⁺-μc's in other rRNAs including the bacterial 16S rRNA, and the P4–P6 domain of the tetrahymena Group I intron ribozyme. It appears that Mg²⁺-μc's are a primeval motif, with pivotal roles in RNA folding, function and evolution.     

Platinum cross-linking of adenines and guanines on the quadruplex structures of the AG3(T2AG3)3 and (T2AG3)4 human telomere sequences in Na+ and K+ solutions

The quadruplex structures of the human telomere sequences AG₃(T₂AG₃)₃ I and (T₂AG₃)₄ II were investigated in the presence of Na⁺ and K⁺ ions, through the cross-linking of adenines and guanines by the cis- and trans-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ complexes 1 and 2. The bases involved in chelation of the cis- and trans-Pt(NH₃)₂ moieties were identified by chemical and 3′-exonuclease digestions of the products isolated after denaturing gel electrophoresis. These are the four adenines of each sequence and four out of the 12 guanines. Two largely different structures have been reported for I: A from NMR data in Na⁺ solution and B from X-ray data of a K⁺-containing crystal. Structure A alone agrees with our conclusions about the formation of the A1–G10, A13–G22, A1–A13 platinum chelates at the top of the quadruplex and A7–A19, G4–A19 and A7–G20 at the bottom, whether the Na⁺ or K⁺ ion is present. At variance with a recent proposal that structures A and B could be the major species in Na⁺ and K⁺ solutions, respectively, our results suggest that structure A exists predominantly in the presence of both ions. They also suggest that covalent platinum cross-linking of a human telomere sequence could be used to inhibit telomerase.     

The phosphate clamp: sequence selective nucleic acid binding profiles and conformational induction of endonuclease inhibition by cationic Triplatin complexes

The substitution-inert polynuclear platinum(II) complex (PPC) series, [{trans-Pt(NH₃)₂(NH₂(CH₂)_nNH₃)}₂-μ-(trans-Pt(NH₃)₂(NH₂(CH₂)_nNH₂)₂}](NO₃)₈, where n = 5 (AH78P), 6 (AH78 TriplatinNC) and 7 (AH78H), are potent non-covalent DNA binding agents where nucleic acid recognition is achieved through use of the ‘phosphate clamp'' where the square-planar tetra-am(m)ine Pt(II) coordination units all form bidentate N–O–N complexes through hydrogen bonding with phosphate oxygens. The modular nature of PPC–DNA interactions results in high affinity for calf thymus DNA (K_app ∼5 × 10⁷ M⁻¹). The phosphate clamp–DNA interactions result in condensation of superhelical and B-DNA, displacement of intercalated ethidium bromide and facilitate cooperative binding of Hoechst 33258 at the minor groove. The effect of linker chain length on DNA conformational changes was examined and the pentane-bridged complex, AH78P, was optimal for condensing DNA with results in the nanomolar region. Analysis of binding affinity and conformational changes for sequence-specific oligonucleotides by ITC, dialysis, ICP-MS, CD and 2D-¹H NMR experiments indicate that two limiting modes of phosphate clamp binding can be distinguished through their conformational changes and strongly suggest that DNA condensation is driven by minor-groove spanning. Triplatin-DNA binding prevents endonuclease activity by type II restriction enzymes BamHI, EcoRI and SalI, and inhibition was confirmed through the development of an on-chip microfluidic protocol.     

Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH₂-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γ_A/γ_A isoform of fibrin(ogen) and the γ_A/γ′ variant with an extended γ-chain. Thrombin binds to the γ′-chain and forms a higher affinity interaction with γ_A/γ′-fibrin(ogen) than γ_A/γ_A-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (K_d values of about 0.5 μm) even though it does not interact with the γ′-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γ_A/γ_A-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a K_d value of 2–5 μm, the α(17–51) and Bβ(1–42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.     

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase

Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants S63E, S68E, and S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control α1β1, FXYD1 reduces V_max and turnover rate and raises K_0.5Na. The phosphomimetic mutants reverse these effects and reduce K_0.5Na below control K_0.5Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K_0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E₂(2K)ATP → E₁(3Na)ATP but does not affect 3NaE₁P → E₂P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60–70) docks between the αN and αP domains in the E₂ conformation, but docking is weaker in E₁ (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na⁺-selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies.     

Cyclic AMP- and (Rp)-cAMPS-induced Conformational Changes in a Complex of the Catalytic and Regulatory (RIα) Subunits of Cyclic AMP-dependent Protein Kinase

We took a discovery approach to explore the actions of cAMP and two of its analogs, one a cAMP mimic ((S_p)-adenosine cyclic 3′:5′-monophosphorothioate ((S_p)-cAMPS)) and the other a diastereoisomeric antagonist ((R_p)-cAMPS), on a model system of the type Iα cyclic AMP-dependent protein kinase holoenzyme, RIα(91–244)·C-subunit, by using fluorescence spectroscopy and amide H/²H exchange mass spectrometry. Specifically, for the fluorescence experiments, fluorescein maleimide was conjugated to three cysteine single residue substitution mutants, R92C, T104C, and R239C, of RIα(91–244), and the effects of cAMP, (S_p)-cAMPS, and (R_p)-cAMPS on the kinetics of R-C binding and the time-resolved anisotropy of the reporter group at each conjugation site were measured. For the amide exchange experiments, ESI-TOF mass spectrometry with pepsin proteolytic fragmentation was used to assess the effects of (R_p)-cAMPS on amide exchange of the RIα(91–244)·C-subunit complex. We found that cAMP and its mimic perturbed at least parts of the C-subunit interaction Sites 2 and 3 but probably not Site 1 via reduced interactions of the linker region and αC of RIα(91–244). Surprisingly, (R_p)-cAMPS not only increased the affinity of RIα(91–244) toward the C-subunit by 5-fold but also produced long range effects that propagated through both the C- and R-subunits to produce limited unfolding and/or enhanced conformational flexibility. This combination of effects is consistent with (R_p)-cAMPS acting by enhancing the internal entropy of the R·C complex. Finally, the (R_p)-cAMPS-induced increase in affinity of RIα(91–244) toward the C-subunit indicates that (R_p)-cAMPS is better described as an inverse agonist because it decreases the fractional dissociation of the cyclic AMP-dependent protein kinase holoenzyme and in turn its basal activity.Cyclic AMP-dependent protein kinase (PKA)¹ plays a crucial role in a plethora of cellular functions. All isoforms of PKA are composed of two catalytic (C) subunits and homodimeric regulatory (R) subunits (1–3). As the name implies, cAMP is a major PKA regulator (4). Much progress has been made in the last decade in delineating the molecular basis of action of cAMP. An important tactic in this endeavor has been through the comparison of the effects of cAMP with those of two phosphorothioate cAMP analogs: (S_p)-cAMPS (a cAMP mimic) and (R_p)-cAMPS (an antagonist and a diastereoisomer of (S_p)-cAMPS). Although the importance of geometry of the sulfur substitution is critical in determining the pharmacological properties of the two phosphorothioate cAMP analogs, the molecular basis for this behavior is not fully understood. To date, these comparisons have only been made using either wild-type or truncated mutants of the type Iα regulatory subunit (RIα) that are free in solution, not complexed to the C-subunit. X-ray spectroscopic examination of ligand-bound RIα(92–379) complexes reveals few differences between ligand-bound complexes, but the (R_p)-cAMPS complex is structurally “looser” with higher thermal factors than complexes formed with either cAMP or (S_p)-cAMPS (5). This is consistent with the observation that both cAMP and (S_p)-cAMPS, but not (R_p)-cAMPS, raise the urea concentration required for wild-type RIα unfolding (6). Further insight into the structural basis of cAMP action stems from NMR spectroscopic comparison of the effects of (R_p)-cAMPS, cAMP, and (S_p)-cAMPS on chemical shifts and ¹⁵N relaxation of the RIα(119–244) mutant (7). In addition to producing fewer significant chemical shift changes than either cAMP or (S_p)-cAMPS, (R_p)-cAMPS binding is associated with enhanced millisecond to microsecond time scale backbone motions of a β-turn (β2,3 loop) and around the phosphate-binding cassette (PBC) (7).Further insight into the molecular basis of actions of cAMP and its analogs should come from the analysis of ligand-bound R·C complexes. Unfortunately, the large size of even the heterodimeric R·C complex (∼95 kDa) and the difficulty of preparing (R_p)-cAMPS·R·C-subunit crystals currently preclude the use of both NMR spectroscopy and x-ray crystallography. Consequently, we took two alternative lower resolution approaches to this issue. One approach involves the use of site-directed labeling combined with fluorescence spectroscopy to examine both the effects of cAMP and its analogs on R-C subunit binding kinetics and on the conformational dynamics of RIα(91–244). RIα(91–244) includes the “A” cyclic nucleotide binding (CNB) domain, the pseudosubstrate, and linker domains and represents the minimal segments necessary for high affinity C-subunit binding (Fig. 1) (8). The other approach involves an examination of the effects of cAMP and its analogs on solvent exposure/conformational flexibility of RIα(91–244)·C-subunit complex using H/²H amide exchange measured with a combination of mass spectrometry (ESI-Q-TOF) and proteolytic fragmentation. In the first approach, fluorescein maleimide (FM) was conjugated to three cysteine substitution mutants with the substitution sites located near or within the pseudosubstrate sequence, the linker domain, or αC (R92C, T104C, and R239C, respectively) of RIα(91–244) (Fig. 1). The time-resolved fluorescence anisotropy results suggest that cAMP and (S_p)-cAMPS reduce the interaction of the RIα linker domain and αC with the two peripheral R-C interaction sites on the C-subunit (so-called Sites 2 and 3) without affecting the interaction of the pseudosubstrate sequence with the active site cleft (so-called Site 1). Because of limitations of the amide H/²H exchange experiments, only the effects of (R_p)-cAMPS on H/²H amide exchange in RIα(91–244)·C-subunit complex could be investigated. The results showed that (R_p)-cAMPS induces a relatively widespread increase in amide exchange, indicating limited unfolding and/or enhanced conformational flexibility that is propagated almost globally through the C-subunit and, at least, part of RIα. These conformational changes were accompanied by a 5-fold increase in the affinity of RIα(91–244) toward C-subunit, suggesting that, at least, some of the (R_p)-cAMPS effects are mediated by an increase in internal entropy. Finally, the (R_p)-cAMPS-induced increase in R-C affinity indicates that (R_p)-cAMPS is better described as an inverse agonist because the basal activity of the PKA holoenzyme should be decreased by (R_p)-cAMPS.Open in a separate windowFig. 1.Overview of PKA structure and cAMP analogs. A, domain organization of RIα showing the domain boundaries of RIα(91–244) where the pseudosubstrate in green is connected to CNB-A domain in blue by a linker segment. B, structure of RIα(91–244) in the C-subunit-bound conformation (Protein Data Bank code 1U7E (23)) showing the pseudosubstrate in green, linker in yellow, and helical subdomain comprising helices αN, αA, αB, and αC in blue and β-subdomain in tan. The PBC is in red. C, structure of the C·RIα(91–244) holoenzyme showing the C-subunit in tan and RIα(91–244) in blue. Sites for introduction of cysteines by site-directed mutagenesis are represented by red circles. The cAMP binding site (PBC) is in red. D, structure of cAMP showing the 2′-OH group and 3′–5′ phosphodiester bond. The exocyclic oxygens upon replacement with sulfur atoms to generate the (S_p)-cAMPS and (R_p)-cAMPS diastereomers are highlighted.     

Aromatic N versus aromatic F: bioisosterism discovered in RNA base pairing interactions leads to a novel class of universal base analogs

The thermodynamics of base pairing is of fundamental importance. Fluorinated base analogs are valuable tools for investigating pairing interactions. To understand the influence of direct base–base interactions in relation to the role of water, pairing free energies between natural nucleobases and fluorinated analogs are estimated by potential of mean force calculations. Compared to pairing of AU and GC, pairing involving fluorinated analogs is unfavorable by 0.5–1.0 kcal mol⁻¹. Decomposing the pairing free energies into enthalpic and entropic contributions reveals fundamental differences for Watson–Crick pairs compared to pairs involving fluorinated analogs. These differences originate from direct base–base interactions and contributions of water. Pairing free energies of fluorinated base analogs with natural bases are less unfavorable by 0.5–1.0 kcal mol⁻¹ compared to non-fluorinated analogs. This is attributed to stabilizing C–F^…H–N dipolar interactions and stronger N^…H–C hydrogen bonds, demonstrating direct and indirect influences of fluorine. 7-methyl-7H-purine and its 9-deaza analog (Z) have been suggested as members of a new class of non-fluorinated base analogs. Z is found to be the least destabilizing universal base in the context of RNA known to date. This is the first experimental evidence for nitrogen-containing heterocylces as bioisosteres of aromatic rings bearing fluorine atoms.     

Hydrodynamic Determinants of Cell Necrosis and Molecular Delivery Produced by Pulsed Laser Microbeam Irradiation of Adherent Cells

Time-resolved imaging, fluorescence microscopy, and hydrodynamic modeling were used to examine cell lysis and molecular delivery produced by picosecond and nanosecond pulsed laser microbeam irradiation in adherent cell cultures. Pulsed laser microbeam radiation at λ = 532 nm was delivered to confluent monolayers of PtK₂ cells via a 40×, 0.8 NA microscope objective. Using laser microbeam pulse durations of 180–1100 ps and pulse energies of 0.5–10.5 μJ, we examined the resulting plasma formation and cavitation bubble dynamics that lead to laser-induced cell lysis, necrosis, and molecular delivery. The cavitation bubble dynamics are imaged at times of 0.5 ns to 50 μs after the pulsed laser microbeam irradiation, and fluorescence assays assess the resulting cell viability and molecular delivery of 3 kDa dextran molecules. Reductions in both the threshold laser microbeam pulse energy for plasma formation and the cavitation bubble energy are observed with decreasing pulse duration. These energy reductions provide for increased precision of laser-based cellular manipulation including cell lysis, cell necrosis, and molecular delivery. Hydrodynamic analysis reveals critical values for the shear-stress impulse generated by the cavitation bubble dynamics governs the location and spatial extent of cell necrosis and molecular delivery independent of pulse duration and pulse energy. Specifically, cellular exposure to a shear-stress impulse J?0.1 Pa s ensures cell lysis or necrosis, whereas exposures in the range of 0.035?J?0.1 Pa s preserve cell viability while also enabling molecular delivery of 3 kDa dextran. Exposure to shear-stress impulses of J?0.035 Pa s leaves the cells unaffected. Hydrodynamic analysis of these data, combined with data from studies of 6 ns microbeam irradiation, demonstrates the primacy of shear-stress impulse in determining cellular outcome resulting from pulsed laser microbeam irradiation spanning a nearly two-orders-of-magnitude range of pulse energy and pulse duration. These results provide a mechanistic foundation and design strategy applicable to a broad range of laser-based cellular manipulation procedures.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Experimental investigation of X-ray lasing in a capillary argon discharge

Results are presented from experiments on the laser generation of X-ray radiation at the wavelength λ=469 ? (ε=26.4 eV) on the 3p(J=0)−3s(J=1) transition of Ne-like Ar ions. Experiments were carried out on the SIGNAL electrophysical facility with a 3.1-mm-diameter 157-mm-long Al₂O₃ ceramic capillary filled with argon at a pressure of 0.2–1.0 Torr. The discharge current amplitude was I ∼ 25–40 kA, the current rise rate being dI/dt ∼ 10¹² A/s. By a vacuum X-ray diode tuned to detect X-ray photons with energies in the range 10–40 eV, laser pulses with a duration of t ₁ ∼ 1 ns and maximum energy of E _1,max ∼ 1 μJ were recorded. The pulses were generated 35 ns after the discharge current was switched on. The line spectra in the wavelength range of 150–500 ? showed the bright λ=469 ? line. The angular divergence of the generated X-ray laser beam was estimated to be Δϑ ∼ 2 mrad. Original Russian Text ? O.N. Gilev, V.I. Afonin, V.I. Ostashev, V.Yu. Politov, A.M. Gafarov, A.L. Zapysov, A.V. Andriyash, é.P. Magda, L.N. Shamraev, A.A. Safronov, A.V. Komissarov, N.A. Khavronin, N.A. Pkhaĭko, L.V. Antonova, L.N. Shushlebin, 2006, published in Fizika Plazmy, 2006, Vol. 32, No. 2, pp. 160–165.     

Heparan Sulfate Containing Unsubstituted Glucosamine Residues: BIOSYNTHESIS AND HEPARANASE-INHIBITORY ACTIVITY*

Degradation of heparan sulfate (HS) in the extracellular matrix by heparanase is linked to the processes of tumor invasion and metastasis. Thus, a heparanase inhibitor can be a potential anticancer drug. Because HS with unsubstituted glucosamine residues accumulates in heparanase-expressing breast cancer cells, we assumed that these HS structures are resistant to heparanase and can therefore be utilized as a heparanase inhibitor. As expected, chemically synthetic HS-tetrasaccharides containing unsubstituted glucosamine residues, GlcAβ1–4GlcNH₃⁺(6-O-sulfate)α1–4GlcAβ1–4GlcNH₃⁺(6-O-sulfate), inhibited heparanase activity and suppressed invasion of breast cancer cells in vitro. Bifunctional NDST-1 (N-deacetylase/N-sulfotransferase-1) catalyzes the modification of N-acetylglucosamine residues within HS chains, and the balance of N-deacetylase and N-sulfotransferase activities of NDST-1 is thought to be a determinant of the generation of unsubstituted glucosamine. We also report here that EXTL3 (exostosin-like 3) controls N-sulfotransferase activity of NDST-1 by forming a complex with NDST-1 and contributes to generation of unsubstituted glucosamine residues.     

Structures and excitation energies of ozone-water clusters O3(H2O)n (n = 1-4)

Hybrid density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations have been carried out for ozone-water clusters O₃(H₂O)_n (n = 1-4) in order to obtain hydration effects on the absorption spectrum of ozone. The first water molecule in n = 1 is bound to the ozone molecule by an oxygen orientation form in which the oxygen atom of H₂O orients the central oxygen atom of O₃. In n = 2, the water dimer is bound to O₃ and then the cyclic structure is formed as the most stable structure. For n = 3 (or n = 4), the cyclic water trimer (or tetramer) is bound by a hydrogen bond to the ozone molecule. The TD-DFT calculations of O₃(H₂O)_n (n = 0-4) show that the first and second excitation energies of O₃ are blue-shifted by the interaction with the water clusters. The magnitude of the spectral shift is largest in n = 2, and the shifts of the excitation energies are +0.07 eV for S₁ and +0.13 eV for S₂ states. In addition to the spectral shifts (S₁ and S₂ states), it is suggested that a charge-transfer band is appeared as a low-lying excited state above the S₁ and S₂ states. The origin of the spectrum shifts was discussed on the basis of theoretical results.     

Electron attachment-induced DNA single-strand breaks at the pyrimidine sites

To elucidate the contribution of pyrimidine in DNA strand breaks caused by low-energy electrons (LEEs), theoretical investigations of the LEE attachment-induced C_3′–O_3′, and C_5′–O_5′ σ bond as well as N-glycosidic bond breaking of 2′-deoxycytidine-3′,5′-diphosphate and 2′-deoxythymidine-3′,5′-diphosphate were performed using the B3LYP/DZP++ approach. The base-centered radical anions are electronically stable enough to assure that either the C–O or glycosidic bond breaking processes might compete with the electron detachment and yield corresponding radical fragments and anions. In the gas phase, the computed glycosidic bond breaking activation energy (24.1 kcal/mol) excludes the base release pathway. The low-energy barrier for the C_3′–O_3′ σ bond cleavage process (∼6.0 kcal/mol for both cytidine and thymidine) suggests that this reaction pathway is the most favorable one as compared to other possible pathways. On the other hand, the relatively low activation energy barrier (∼14 kcal/mol) for the C_5′–O_5′ σ bond cleavage process indicates that this bond breaking pathway could be possible, especially when the incident electrons have relatively high energy (a few electronvolts). The presence of the polarizable medium greatly increases the activation energies of either C–O σ bond cleavage processes or the N-glycosidic bond breaking process. The only possible pathway that dominates the LEE-induced DNA single strands in the presence of the polarizable surroundings (such as in an aqueous solution) is the C_3′–O_3′ σ bond cleavage (the relatively low activation energy barrier, ∼13.4 kcal/mol, has been predicted through a polarizable continuum model investigation). The qualitative agreement between the ratio for the bond breaks of C_5′–O_5′, C_3′–O_3′ and N-glycosidic bonds observed in the experiment of oligonucleotide tetramer CGAT and the theoretical sequence of the bond breaking reaction pathways have been found. This consistency between the theoretical predictions and the experimental observations provides strong supportive evidences for the base-centered radical anion mechanism of the LEE-induced single-strand bond breaking around the pyrimidine sites of the DNA single strands.     

Yeast telomerase appears to frequently copy the entire template in vivo

Telomeres derived from the same formation event in wild type strains of Saccharomyces cerevisiae possess the same, precise TG_1–3 sequence for the most internal ~100 bp of the 250–350 bp TG_1–3 repeats. The conservation of this internal domain is thought to reflect the fact that telomere lengthening and shortening, and thus alteration of the precise TG_1–3 sequence, is confined to the terminal region of the telomere. The internal domains of telomeres from yku70Δ and tel1Δ mutants, whose entire telomeres are only ~100 bp, were examined by analyzing 5.1 kb of cloned TG_1–3 sequences from telomeres formed during transformation of wild type, yku70Δ and tel1Δ cells. The internal domains were 97–137 bp in wild type cells, 27–36 bp in yku70Δ cells and 7–9 bp in tel1Δ cells. These data suggest that the majority of the tel1Δ cell TG_1–3 repeats may be resynthesized during shortening and lengthening reactions while a portion of the yku70Δ cell telomeres are protected. TG_1–3 sequences are synthesized by telomerase repeatedly copying an internal RNA template, which introduces a sequence bias into TG_1–3 repeats. Analysis of in vivo-derived telomeres revealed that of the many possible high affinity binding sites for the telomere protein Rap1p in TG_1–3 repeats, only those consistent with telomere hybridization to the ACACAC in the 3′-region of the telomerase RNA template followed by copying of most of the template were present. Copies of the telomerase RNA template made up 40–60% of the TG_1–3 sequences from each strain and could be found in long, tandem repeats. The data suggest that in vivo yeast telomerase frequently allows telomeres to hybridize to the 3′-region of RNA template and copy most of it prior to dissociation, or that in vivo telomere processing events result in the production of TG_1–3 sequences that mimic this process.     

Temperature Sensitivity of Microbial Respiration of Fine Root Litter in a Temperate Broad-Leaved Forest

The microbial decomposition respiration of plant litter generates a major CO₂ efflux from terrestrial ecosystems that plays a critical role in the regulation of carbon cycling on regional and global scales. However, the respiration from root litter decomposition and its sensitivity to temperature changes are unclear in current models of carbon turnover in forest soils. Thus, we examined seasonal changes in the temperature sensitivity and decomposition rates of fine root litter of two diameter classes (0–0.5 and 0.5–2.0 mm) of Quercus serrata and Ilex pedunculosa in a deciduous broad-leaved forest. During the study period, fine root litter of both diameter classes and species decreased approximately exponentially over time. The Q₁₀ values of microbial respiration rates of root litter for the two classes were 1.59–3.31 and 1.28–6.27 for Q. serrata and 1.36–6.31 and 1.65–5.86 for I. pedunculosa. A significant difference in Q₁₀ was observed between the diameter classes, indicating that root diameter represents the initial substrate quality, which may determine the magnitude of Q₁₀ value of microbial respiration. Changes in these Q₁₀ values were related to seasonal soil temperature patterns; the values were higher in winter than in summer. Moreover, seasonal variations in Q₁₀ were larger during the 2-year decomposition period than the 1-year period. These results showed that the Q₁₀ values of fine root litter of 0–0.5 and 0.5–2.0 mm have been shown to increase with lower temperatures and with the higher recalcitrance pool of the decomposed substrate during 2 years of decomposition. Thus, the temperature sensitivity of microbial respiration in root litter showed distinct patterns according to the decay period and season because of the temperature acclimation and adaptation of the microbial decomposer communities in root litter.     

Water transport in invertebrate peripheral nerve fibers

Osmotic and diffusion permeabilities (P_f and P_d) of invertebrate nerve fibers to tritiated water were measured to determine what water flux studies could reveal about "the nerve membrane" and to directly test the possibility of active transport of water into or out of invertebrate nerve fibers. P_f/P_d ratios for lobster walking leg nerve fibers were found to be about 20 ± 7 at 14°C. P_d measurements were made for squid giant axons at 25°C. and found to yield a value of 4 x 10^–4 cm.^–1 sec.^–1. When combined with the data of D. K. Hill for P_f, a P_f/P_d ratio of 21 ± 5 is obtained. These P_f/P_d ratios correspond to "effective pore radii" of about 16 ± 4 angstrom units, according to theories developed by Koefoed-Johnsen and Ussing and independently by Pappenheimer and his colleagues. Variations of water flux ratios with temperatures were studied and apparent activation energies calculated for both diffusion experiments and osmotic filtration experiments using the Arrhenius equation, and found to be close to 3 to 5 cal. per mole of water transferred. Cyanide (5 x 10^–3 molar) and iodoacetate (1 x 10^–3 molar) poisoned lobster leg nerve fibers showed no appreciable change in diffusion or osmotic filtration water effluxes. Caution in interpreting these proposed channels as simple pores was emphasized, but the possibility that such channels exist and are related to ionic flow is not incompatible with electrophysiological data.     

The C-Terminal Sequence of IFITM1 Regulates Its Anti-HIV-1 Activity

The interferon-inducible transmembrane (IFITM) proteins inhibit a wide range of viruses. We previously reported the inhibition of human immunodeficiency virus type 1 (HIV-1) strain BH10 by human IFITM1, 2 and 3. It is unknown whether other HIV-1 strains are similarly inhibited by IFITMs and whether there exists viral countermeasure to overcome IFITM inhibition. We report here that the HIV-1 NL4-3 strain (HIV-1_NL4-3) is not restricted by IFITM1 and its viral envelope glycoprotein is partly responsible for this insensitivity. However, HIV-1_NL4-3 is profoundly inhibited by an IFITM1 mutant, known as Δ(117–125), which is deleted of 9 amino acids at the C-terminus. In contrast to the wild type IFITM1, which does not affect HIV-1 entry, the Δ(117–125) mutant diminishes HIV-1_NL4-3 entry by 3-fold. This inhibition correlates with the predominant localization of Δ(117–125) to the plasma membrane where HIV-1 entry occurs. In spite of strong conservation of IFITM1 among most species, mouse IFITM1 is 19 amino acids shorter at its C-terminus as compared to human IFITM1 and, like the human IFITM1 mutant Δ(117–125), mouse IFITM1 also inhibits HIV-1 entry. This is the first report illustrating the role of viral envelope protein in overcoming IFITM1 restriction. The results also demonstrate the importance of the C-terminal region of IFITM1 in modulating the antiviral function through controlling protein subcellular localization.     

Refined genetic localization for central core disease

Central core disease (CCO) is an autosomal dominant myopathy clinically distinct from malignant hyperthermia (MHS). In a large kindred in which the gene for CCO is segregating, two-point linkage analysis gave a maximum lod score, between the central core disease locus (CCO) and the ryanodine receptor locus (RYR1), of 11.8, with no recombination. Mutation within RYR1 is responsible for MHS, and RYR1 is also a candidate locus for CCO. A combination of physical mapping using a radiation-induced human-hamster hybrid panel and of multipoint linkage analysis using the Centre d'Etude du Polymorphisme Humain families established the marker order and sex-average map distances (in centimorgans) on the background map as D19S75–(5.2)–D19S9–(3.4)–D19S191–(2.2)–RYR1–(1.7)–D19S190–(1.6)-D19S47–(2.0)–CYP2B. Recombination was observed between CCO and the markers flanking RYR1. These linkage data are consistent with the hypothesis that CCO and RYR1 are allelic. The most likely position for CCO is near RYR1, with a multipoint lod score of 11.4, in 19q13.1 between D19S191 and D19S190, within the same interval as MHS (RYR1).     

Dielectric Dispersion in Ga<Subscript>2</Subscript>S<Subscript>3</Subscript> Thin Films

In this work, the structural, compositional, optical, and dielectric properties of Ga₂S₃ thin films are investigated by means of X-ray diffraction, scanning electron microscopy, energy dispersion X-ray analysis, and ultraviolet—visible light spectrophotometry. The Ga₂S₃ thin films which exhibited amorphous nature in its as grown form are observed to be generally composed of 40.7 % Ga and 59.3 % S atomic content. The direct allowed transitions optical energy bandgap is found to be 2.96 eV. On the other hand, the modeling of the dielectric spectra in the frequency range of 270–1,000 THz, using the modified Drude-Lorentz model for electron-plasmon interactions revealed the electrons scattering time as 1.8 (fs), the electron bounded plasma frequency as ~0.76–0.94 (GHz) and the reduced resonant frequency as 2.20–4.60 ×10¹⁵ (Hz) in the range of 270–753 THz. The corresponding drift mobility of electrons to the terahertz oscillating incident electric field is found to be 7.91 (cm ²/Vs). The values are promising as they nominate the Ga₂S₃ thin films as effective candidates in thin-film transistor and gas sensing technologies.     

Relationship between Sevoflurane Plasma Concentration,Clinical Variables and Bispectral Index Values during Cardiopulmonary Bypass

Background

Anesthetic administration is increasingly guided by electroencephalography (EEG)-based monitoring, such as the bispectral index (BIS). However, during cardiopulmonary bypass (CPB), factors other than the administered hypnotic agents may influence EEG signals, and their effects on BIS values are unknown.

Methods

This report is a secondary analysis of data from a prospective, controlled interventional study comparing the effect of sevoflurane administration guided by BIS monitoring (group Sevo_BIS) and constant administration of sevoflurane (group Sevo_1.8Vol%) during CPB. Sevoflurane plasma concentration (SPC) was measured using gas chromatography. The relationships of BIS to SPC, CPB pump flow, arterial pressure, hematocrit, temperature, time on CPB, and patient characteristics were analysed.

Results

No association was observed between BIS values and SPC in group Sevo_BIS. In group Sevo_1.8Vol%, a 40 μg ml^-1 increase in SPC, which encompassed the entire range of observed values of the SPC in this analysis, was associated with a decrease of 3.6 (95% confidence interval (CI): 1.1–6.1) in BIS values (p = 0.005). Each increase in CPB time of 10 minutes was associated with an increase in BIS values of 0.25 (95%CI: 0.11–0.39, p<0.001). Path analysis revealed that the BIS values of Sevo_BIS patients were 5.3 (95%CI: 3.2–7.5) units higher than those of Sevo_1.8Vol% patients (p<0.001), which was the strongest effect on BIS values. Path analysis revealed a slope of 0.5 (95%CI: 0.3–0.7) BIS units per 1°C body temperature (p<0.001).

Conclusion

BIS monitoring is insensitive to clinically relevant changes in SPC in individual patients during CPB.