Influence of cation–π interactions to the structural stability of prokaryotic and eukaryotic translation elongation factors

We have investigated the role of cation–π interactions on translation elongation factors. In our investigation, an average of four significant cation–π interactions were found, that is, an average of one cation–π interaction per 44 residues in the ten elongation factors were observed. The analysis on the influence of short (<±4), medium (>±4 to <±20) and long (>20) range contacts showed that cation–π interactions are mainly formed by medium and long-range contacts. Arg-Tyr pair was found largest in number but energetic contribution of Arg-Trp pair was found most. Preferred secondary structural conformation analysis of the residues involved in cation–π interaction indicates that the cationic Arg prefers to be in helix and Lys having equal probability for helix and strand, whereas the aromatic Phe and Trp were found mostly in helix while Tyr in strand regions. The cation–π interaction residues involved in these proteins were found highly conserved with 48.86% residues having conservation score of ≥6. Analysis of secondary structure preference of the energetically significant cation–π residues in different solvent accessible range indicates that most of the π residues are found buried or partially buried whereas cationic residues were found mostly at the protein surface. The results presented in this study will be useful for structural stability studies in translation elongation factors.     

Measurement of membrane potentials (ψ) of erythrocytes and white adipocytes by the accumulation of triphenylmethylphosphonium cation

Summary The accumulation of the lipophilic cation, triphenylmethylphosphonium, has been employed to determine the resting membrane potential in human erythrocytes, turkey erythrocytes, and rat white adipocytes. The triphenylmethylphosphonium cation equilibrates rapidly in human erythrocytes in the presence of low concentrations of the hydrophobic anion, tetraphenylborate. Tetraphenylborate does not accelerate the uptake of triphenylmethylphosphonium ion by adipocytes. The cell associatedvs. extracellular distribution of the triphenylmethylphosphonium ion is proportional to changes in membrane potential. The distribution of this ion reflects the membrane potential determining concentration of the ion with dominant permeability in a Nernst fashion. The resting membrane potentials for the human erythrocyte, turkey erythrocyte, and rat white adipocyte were found to be –8.4±1.3, –16.8±1.1, and –58.3±5.0 mV, respectively, values which compare favorably with values obtained by other methods. In addition, changes in membrane potential can be assessed by following triphenylmethylphosphonium uptake without determining the intracellular water space. The method has been successfully applied to a study of hormonally induced changes in membrane potential of rat white adipocytes.     

Protein–ligand interactions: Probing the energetics of a putative cation–π interaction

In order to probe the energetics associated with a putative cation–π interaction, thermodynamic parameters are determined for complex formation between the Grb2 SH2 domain and tripeptide derivatives of RCO–pTyr–Ac6c–Asn wherein the R group is varied to include different alkyl, cycloalkyl, and aryl groups. Although an indole ring is reputed to have the strongest interaction with a guanidinium ion, binding free energies, ΔG°, for derivatives of RCO–pTyr–Ac6c–Asn bearing cyclohexyl and phenyl groups were slightly more favorable than their indolyl analog. Crystallographic analysis of two complexes reveals that test ligands bind in similar poses with the notable exception of the relative orientation and proximity of the phenyl and indolyl rings relative to an arginine residue of the domain. These spatial orientations are consistent with those observed in other cation–π interactions, but there is no net energetic benefit to such an interaction in this biological system. Accordingly, although cation–π interactions are well documented as important noncovalent forces in molecular recognition, the energetics of such interactions may be mitigated by other nonbonded interactions and solvation effects in protein–ligand associations.     

Review of the fauna of Rotatoria,Cladocera, and Copepoda of the basin of the Anadyr’ River

The zooplankton composition is studied in the thermokarst, glacial and meteorite lakes, channels, former riverbeds, and hollows in the basin of Anadyr’. We found 174 taxa: 78, Rotatoria, 55, Cladocera, and 41, Copepoda. The most diverse is the lake fauna: 51 taxa of Rotatoria, 48, Cladocera, and 37, Copepoda. The thermokarst Lake Maiorskoe hosts 68 taxa: 31, Rotatoria, 14, Cladocera, and 23, Copepoda, wheras the cold ultraoligotrophic Lake El’gygytgyn features only one species of Cyclop of the group scutifer Cyclops neymanae Strel., and Rotatoria and Cladocera are present as allochtonous forms. The Copepoda illustrate the relations of the Anadyr’ fauna with those of Europe, North America, and Japan.     

Investigation of cation–π interactions in sugar-binding proteins

Cation–π interaction is a non-covalent binding force that plays a significant role in protein stability and drug–receptor interactions. In this work, we have investigated the structural role of cation–π interactions in sugar-binding proteins (SBPs). We observed 212 cation–π interactions in 53 proteins out of 59 SBPs in dataset. There is an average one energetically significant cation–π interaction for every 66 residues in SBPs. In addition, Arg is highly preferred to form cation–π interactions, and the average energy of Arg-Trp is high among six pairs. Long-range interactions are predominant in the analyzed cation–π interactions. Comparatively, all interaction pairs favor to accommodate in strand conformations. The analysis of solvent accessible area indicates that most of the aromatic residues are found on buried or partially buried whereas cationic residues were found mostly on the exposed regions of protein. The cation–π interactions forming residues were found that around 43% of cation–π residues had highly conserved with the conservation score ≥6. Almost cationic and π-residues equally share in the stabilization center. Sugar-binding site analysis in available complexes showed that the frequency of Trp and Arg is high, suggesting the potential role of these two residues in the interactions between proteins and sugar molecules. Our observations in this study could help to further understand the structural stability of SBPs.     

Molecular orbital study of the hydroxylation of benzene and monofluorobenzene catalysed by iron-oxo porphyrin π cation radical complexes

 The reaction mechanism for the hydroxylation of benzene and monofluorobenzene, catalysed by a ferryl-oxo porphyrin cation radical complex (compound) is described by electronic structure calculations in local spin density approximation. The active site of the enzyme is modelled as a six-coordinated (Por⁺)Fe(IV)O a_2u complex with imidazole or H₃CS^– as the axial ligand. The substrates under study are benzene and fluorobenzene, with the site of attack in para, meta and ortho position with respect to F. Two reaction pathways are investigated, with direct oxygen attack leading to a tetrahedral intermediate and arene oxide formation as a primary reaction step. The calculations show that the arene oxide pathway is distinctly less probable, that hydroxylation by an H₃CS^––coordinated complex is energetically favoured compared with imidazole, and that the para position with respect to F is the preferred site for hydroxylation. A partial electron transfer from the substrate to the porphyrin during the reaction is obtained in all cases. The resulting charge distribution and spin density of the substrates reveal the transition state as a combination of a cation and a radical σ-adduct intermediate with slightly more radical character in the case of H₃CS^– as axial ligand. A detailed analysis of the orbital interactions along the reaction pathway yields basically different mechanisms for the modes of substrate–porphyrin electron transfer and rupture of the Fe–O bond. In the imidazole-coordinated complex an antibonding π*(Fe–O) orbital is populated, whereas in the H₃CS^––coordinated system a shift of electron density occurs from the Fe–O bond region into the Fe–S bond. Received: 1 July 1995 / Accepted: 18 December 1995     

Interactions between alkaline earth cations and oxo ligands. DFT study of the affinity of the Mg²+ cation for phosphoryl ligands

DFT (B3LYP/6-31+G(d)) calculations of Mg²⁺ affinities for a set of phosphoryl ligands were performed. Two types of ligands were studied: a set of trivalent [O = P(R)] and a set of pentavalent phosphoryl ligands [O = P(R)₃] (R = H, F, Cl, Br, OH, OCH₃, CH₃, CN, NH₂ and NO₂), with R either bound directly to the phosphorus atom or to the para position of a phenyl ring. The affinity of the Mg²⁺ cation for the ligands was quantified by means of the enthalpy for the substitution of one water molecule in the [Mg(H₂O)₆]²⁺ complex for a ligand. The enthalpy of substitution was correlated with electronic and geometric parameters. Electron-donor groups increase the interaction between the cation and the ligand, while electron-acceptor groups decrease the interaction enthalpy.     

D-glucose interaction with uranium ion. Synthesis,spectroscopic and structural characterization of uranylglucose adducts and the effect of metal cation binding on the sugar anomeric structures

The interaction of D-glucose with hydrated uranyl salts has been investigated in solution and solid adducts of the type UO₂(D-glucose)X₂·2H₂O, where X = Cl⁻, Br⁻, NO_3⁻ and 0.5 SO_4²⁻ have been isolated. These adducts are characterized by means of FT-IR, ¹H NMR and molar conductivity measurements.Spectroscopic evidence suggested that UO_2²⁺ cation could be bonded to one D-glucose molecule (possibly through O(1)H and O(2)H hydroxyl groups) and to two H₂O, resulting in six-coordination around the uranium ion.The strong sugar H-bonding network is perturbed, on metal ion interaction and the D-glucose α-anomeric structure is favoured, upon uranyl cation coordination.     

Characterization of the Fourth α Isoform of the Na,K-ATPase

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K _D values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na⁺ and K⁺. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis. Received: 4 December 1998/Revised: 1 February 1999     

Chitinozoan zonation of the Late Ordovician and the Early Silurian of the island of Anticosti, Québec, Canada

A reappraisal of chitinozoan distribution across the Ordovician-Silurian boundary on the Island of Anticosti has led to the recognition of a new zone, the Ancyrochitina ellisbayensis biozone, in the uppermost part of the Ellis Bay Formation. This biozone lies between the well defined Upper Ordovician Spinachitina taugourdeaui biozone and the lowest Silurian (Rhuddanian) Plectochitina nodifera biozone of the Becscie Formation. The occurrence of such diagnostic species as P. nodifera, Belonechitina postrobusta, Conochitina electa and Ancyrochitina ramosaspina in the Lower Silurian of Anticosti points to a close similarity to faunas in Estonia and north Latvia and indicates an age ranging from the Parakidograptus acuminatus to the Coronograptus cyphus in terms of graptolite zones. The chitinozoan biozonation harmonizes with that based on conodonts and, to a lesser extent, with the known graptolite faunal succession. Five new species: Ancyrochitina ellisbayensis sp. nov., Clathrochitina postconcinna sp. nov., Conochitina gunriveris sp. nov., Clathrochitina perexilis sp. nov., Bursachitina basiconcava sp. nov. and three species in open nomenclature are described.     

Kinetics and mechanism of the aquation of the trinuclear cation, [μ3-oxo-triaqua-hexakis(acetato)tris(iron(III))]in perchloric acid media

The aquation of the title complex cation in aqueous perchloric acid proceeded via two steps, both postulated to be the proton attack on the oxygen atom which binds the acetate ligand to the metal centre, followed by Fe-O bond cleavage. This was followed by rapid decomposition to produce aqueous iron(III) and acetate ions. The first-order rate constants for the first and second steps at 25 °C are: k₁ = (4.16 ± 0.58) × 10⁻² s⁻¹ and k₂ = (2.09 ± 0.42) × 10⁻³ s⁻¹, respectively, and their corresponding activation parameters are . The spontaneous hydrolysis rate constants for the first and second steps were also determined at 25 °C and ionic strength of 1 mol dm⁻³ and they are k₀ = (3.10 ± 0.82) × 10⁻³ s⁻¹ and , respectively. The corresponding activation parameters are .     

Effects of the ΔF508 mutation on the structure,function, and folding of the first nucleotide-binding domain of CFTR

The fatal autosomal recessive disease cystic fibrosis (CF) is caused by mutations in the gene which encodes the cystic fibrosis transmembrane conductance regulator (CFTR). Many of these disease-causing mutations, including the deletion of F508 (F508) which accounts for approximately 70% of the disease alleles, occur in one of the two consensus nucleotide binding sequences. Peptide studies have directly demonstrated that the N-terminal nucleotide binding sequences bind adenine nucleotides. Structurally, circular dichroism spectropolarimetry indicates that this region of CFTR assumes a -stranded structure in solution. The F508 mutation causes a diminution in the amount of -stranded structure and a concomitant increase in the amount of random coil structure present, indicating that either the mutant peptide has a different native structure or that the conformational equilibrium is shifted toward a more disordered form. Furthermore, the mutant peptide is more sensitive to denaturation, indicating that F508 is a stability, or protein-folding mutant. Here we review these results and discuss their implications for interpreting the behavior of F508in situ and for the rational design of new CF drugs.     

Absorption of the biomimetic chromium cation triaqua-μ3-oxo-μ-hexapropionatotrichromium(III) in rats

The cation [Cr₃O(O₂CCH₂CH₃)₆(H₂O)₃]⁺ has been shown in vitro to mimic to the oligopeptide chromodulin’s ability to stimulate the tyrosine kinase activity of insulin receptor and shown in healthy and type 2 diabetic model rats to increase insulin sensitivity and decrease plasma total and low-density lipoprotein cholesterol and triglycerides concentrations. However, the degree to which the complex is absorbed after gavage administration to rats had not been previously determined. The biomimetic cation at nutritional supplement levels is absorbed with greater than 60% efficiency, and at pharmacological levels, it is absorbed with greater than 40% efficiency, an order of magnitude greater absorption than that of CrCl₃, Cr nicotinate, or Cr picolinate, currently marketed nutritional supplements. The difference in degree of absorption is readily explained by the stability and solubility of the cation.     

Cloning,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

SYBR Green Ⅰ-based product-enhanced reverse transcriptase assay for quantification of retroviral PFV and detection of the divalent cation preference of PFV RT

Dear Editor, Prototype foamy virus (PFV) belongs to the genus Spumavirus in the Spumaretrovirinae subfamily of Retroviridae.Although PFV and HIV have much in common,research into PFV has lagged far behind that into HIV,as PFV appeared to be non-pathogenic both in accidentally infected humans and in experimentally infected animals.In recent decades,however,more attention has been focused on PFV because it seems to be a promising candidate vector for gene therapy in clinical applications.Compared with other retroviral vectors,vectors derived from PFV appear to be safer (Sastry L,et al.,2005).In addition,it was found that PFV vectors are efficient vehicles for stable gene transfer in proliferating cells,and that the transferred genes are able to achieve stable integration and long-term expression in the target cells.     

Blood-to-brain influx transport of nicotine at the rat blood–brain barrier: Involvement of a pyrilamine-sensitive organic cation transport process

Nicotine is the most potent neural pharmacological alkaloid in tobacco, and the modulation of nicotine concentration in the brain is important for smoking cessation therapy. The purpose of this study was to elucidate the net flux of nicotine transport across the blood–brain barrier (BBB) and the major contributor to nicotine transport in the BBB. The in vivo brain-to-blood clearance was determined by a combination of the rat brain efflux index method and a rat brain slice uptake study, and the blood-to-brain transport of nicotine was evaluated by in vivo vascular injection in rats and a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13 cells) as an in vitro model of the rat BBB. The blood-to-brain nicotine influx clearance was obtained by integration plot analysis as 272 μL/(min g brain), and this value was twofold greater than the brain-to-blood efflux clearance (137 μL/(min g brain)). Thus, it is suggested that the net flux of nicotine transport across the BBB is dominated by blood-to-brain influx transport. In vivo blood-to-brain nicotine transport was inhibited by pyrilamine. [³H]Nicotine uptake by TR-BBB13 cells exhibited time-, temperature-, and concentration-dependence with a K_m value of 92 μM. Pyrilamine competitively inhibited nicotine uptake by TR-BBB13 cells with a K_i value of 15 μM, whereas substrates and inhibitors of organic cation transporters had little effect. These results suggest that pyrilamine-sensitive organic cation transport process(es) mediate blood-to-brain influx transport of nicotine at the BBB, and this is expected to play an important role in regulating nicotine-induced neural responses.