Transfer of chirality by dithiophosphate ligands and chiral discrimination in the stereoselective formation of square-planar Ni(II) complexes

In the formation reaction of Ni(2+) with the chiral racemic ligand, (R)(R)bdtp(-)/(S)(S)bdtp(-), bdtp(-) = [SSPOCH)CH(3))CH(CH(3))O](-), cyclo- O,O'-[1,2-dimethylethylene] dithiophosphato ion, the meso-complex Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)-bdtp] is stereoselectively produced. The meso-complex was compared with the enantiopure crystals of (+)(589)Ni[(R)(R)(lambda)bdtp](2) or (-)(589)Ni[(S)(S)(delta)bdtp](2), as well as racemic crystals, rac-(+/-)Ni[bdtp](2), which were prepared from the solution containing the two enantiomers in a 1:1 ratio. Dissociation constants in solutions indicate different stability of the meso and enantiopure complexes depending on the solvent, whereas a more efficient crystal packing, weak H-bonding, and nonbonding interactions contribute to stabilization of the meso-species over the racemic one. Molecular structures show that the outer five-membered ligand ring adopts the half-chair conformation C(2) with either the lambda or the delta chirality and the methyl groups are in equatorial (e) positions. Enantiopure ligands of (+)(589)Ni[(R)(R)(lambda)bdtp](2) and (-)(589)Ni[(S)(S)(delta)bdtp](2) induce chirality into the symmetric SSNiSS chromophore with slightly helical distortion. Thus, their CD spectra exhibit weak negative or positive Cotton effects at 662 nm. CD spectra in L(+)- and D(-)diethyltartrate of the meso-complex and racemic crystal, rac-(+/-)Ni[bdtp](2), exhibit different weak Cotton effects of opposite sign. Complexes dissociate in methanol; rac-(+/-)Ni[bdtp](2) in methanol undergoes a crystallization-induced second-order asymmetric transformation which finally yields crystals of the meso-Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)bdtp] complex.     

Molecular modeling study on the enantioselective binding of S- and R-ofloxacin to various DNA sequences

The complex formation of S- and R-ofloxacin with the self-complementary oligonucleotides, namely d[ATAGCGCTAT](2), d[GCGATATCGC](2) and d[ATAICICTAT](2), were investigated by the molecular dynamics (MD) simulation. Four starting positions, including two intercalation positions with different insertion directions and two minor groove binding positions, were considered. The total energy of both S- and R-ofloxacin-d[ATAGCGCTAT](2) complex, in which ofloxacin binds in the minor groove of the oligonucleotide, were lower than any intercalation binding mode. For both enantiomers, formation of the complex with GC oligonucleotide is more favorable than AT and IC oligonucleotides. When S- and R-ofloxacin are compared, the S-enantiomer exhibits more favorable total energy and torsion angles in the complex formation. This result is in agreement with the experimental observation [Hwangbo et al., Eur J Pharm Sci 18, 197 (2003)]. In the complex, both enantiomers form two hydrogen bonds: one between the carbonyl group of ofloxacin and the amine group of G16 and the other between the fluorine group and the G6 amine for S-ofloxacin. However, only one hydrogen bond is formed between endocyclic hydrogen atom at the C2 position of adenine and inosine base and carbonyl group of ofloxacin, which may be the reason for the GC preferentiality of ofloxacin.     

Native-like conformations are sampled by partially folded and disordered variants of bovine pancreatic trypsin inhibitor

Partially folded conformational ensembles of bovine pancreatic trypsin inhibitor (BPTI) are accessed by replacing Cys 5, 30, 51, and 55 by alpha-amino-n-butyric acid (Abu) while retaining the disulfide between Cys 14 and 38; the resultant variant is termed [14-38](Abu). Two new analogues with modifications in the beta-turn, P26D27[14-38](Abu) and N26G27K28[14-38](Abu), are compared to partially folded [14-38](Abu), as well as to [R](Abu), the unfolded protein with all six Cys residues replaced by Abu. Structural features of the new analogues of [14-38](Abu) have been determined by circular dichroism (CD), one-dimensional (1)H NMR, and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence experiments. Both analogues are more disordered than the parent [14-38](Abu), but while P26D27[14-38](Abu) has a small population of native-like conformations observed by NMR, no ordered structure is detected for N26G27K28[14-38](Abu). Trypsin inhibition assays were carried out using a modified rat trypsin, C191A/C220A, that minimizes cleavage of unfolded peptides. Both [14-38](Abu) and P26D27[14-38](Abu) significantly inhibit modified trypsin. N26G27K28[14-38](Abu) has low but measurable inhibitor activity, while [R](Abu) has no activity even when in very high molar excess relative to trypsin. ANS fluorescence is enhanced by [14-38](Abu) and by both variants but not by [R](Abu). We conclude that partially folded ensembles of BPTI, even those with little or no CD- or NMR-detectable structure, contain minor populations of native-like conformations. Partially folded [14-38](Abu) and both variants, as well as [R](Abu), have enhanced negative ellipticity in CD spectra acquired in the presence of the osmolyte trimethylamine N-oxide (TMAO). TMAO-induced structure is formed cooperatively, as indicated by thermal unfolding curves. Inhibitor activity as a function of TMAO concentration implies that the osmolyte-induced structure is native-like for [14-38](Abu) and P26D27[14-38](Abu) and is probably native-like for N26G27K28[14-38](Abu). [R](Abu) also shows increased CD-detected structure in the presence of TMAO, but such structure is likely to be collapsed and non-native.     

Density functional studies on the electron affinities of DNA and RNA bases

Influence of basis sets on electron affinities (EAs) of DNA and RNA bases has been investigated using density functional method (B3LYP functional) with different basis sets (6-31G, TZVP and 6-311+ + G**). Effect of some PBE functionals namely, PBEOP, PBELYP and PBEVWN, on EA values of the nucleobases was studied using basis set which predicted the most reliable values with B3LYP functional. Observation of the trends in the values of EA and dipole moment of the molecules enable us to identify the features of a basis set that shows the presence of dipole-bound state of some of the nucleobases. The vertical electron affinities with B3LYP and PBEOP functionals are close to the experimental values. The adiabatic electron affinities of uracil and thymine were found to be positive for basis set with diffuse functions using B3LYP functional. Adenine does not have a stable covalently bound anion at all levels of basis sets and functionals. The sign of adiabatic electron affinity value of cytosine is inconsistent with that of experimental value but in agreement with previous theoretical results. For guanine the adiabatic electron affinity value with 6-311+ + G** basis set was found to be very high as comparison with other two basis sets confirming the formation of mixed covalent-dipole character.     

Study of Completed Archaeal Genomes and Proteomes： Hypothesis of Strong Mutational AT Pressure Existed in Their Common Predecessor

The number of completely sequenced archaeal genomes has been sufficient for a large-scale bioinformatic study.We have conducted analyses for each coding region from 36 archaeal genomes using the original CGS algorithm by calculating the total GC content(G+C),GC content in first,second and third codon positions as well as in fourfold and twofold degenerated sites from third codon positions,levels of arginine codon usage(Arg2:AGA/G;Arg4:CGX),levels of amino acid usage and the entropy of amino acid content distribution.In archaeal genomes with strong GC pressure,arginine is coded preferably by GC-rich Arg4 codons,whereas in most of archaeal genomes with G+C0.6,arginine is coded preferably by AT-rich Arg2 codons.In the genome of Haloquadratum walsbyi,which is closely related to GC-rich archaea,GC content has decreased mostly in third codon positions,while Arg4Arg2 bias still persists.Proteomes of archaeal species carry characteristic amino acid biases:levels of isoleucine and lysine are elevated,while levels of alanine,histidine,glutamine and cytosine are relatively decreased.Numerous genomic and proteomic biases observed can be explained by the hypothesis of previously existed strong mutational AT pressure in the common predecessor of all archaea.     

Empirical relationships between protein structure and carboxyl pKa values in proteins

Relationships between protein structure and ionization of carboxyl groups were investigated in 24 proteins of known structure and for which 115 aspartate and 97 glutamate pK(a) values are known. Mean pK(a) values for aspartates and glutamates are < or = 3.4 (+/-1.0) and 4.1 (+/-0.8), respectively. For aspartates, mean pK(a) values are 3.9 (+/-1.0) and 3.1 (+/-0.9) in acidic (pI < 5) and basic (pI > 8) proteins, respectively, while mean pK(a) values for glutamates are approximately 4.2 for acidic and basic proteins. Burial of carboxyl groups leads to dispersion in pK(a) values: pK(a) values for solvent-exposed groups show narrow distributions while values for buried groups range from < 2 to 6.7. Calculated electrostatic potentials at the carboxyl groups show modest correlations with experimental pK(a) values and these correlations are not improved by including simple surface-area-based terms to account for the effects of desolvation. Mean aspartate pK(a) values decrease with increasing numbers of hydrogen bonds but this is not observed at glutamates. Only 10 pK(a) values are > 5.5 and most are found in active sites or ligand-binding sites. These carboxyl groups are buried and usually accept no more than one hydrogen bond. Aspartates and glutamates at the N-termini of helices have mean pK(a) values of 2.8 (+/-0.5) and 3.4 (+/-0.6), respectively, about 0.6 units less than the overall mean values.     

Low-energy (5-40 eV) electron-stimulated desorption of anions from physisorbed DNA bases

We present the results of experiments on anion desorption from the physisorbed DNA bases adenine, thymine, guanine and cytosine induced by the impact of low-energy (5-40 eV) electrons. Electron bombardment of DNA base films induces ring fragmentation and desorption of H(-), O(-), OH(-), CN(-), OCN(- ) and CH(2)(-) anions through either single or complex multibond dissociation. We designate the variation of the yield of an anion with electron energy as the yield function. Below 15 eV incident electron energy, bond cleavage is controlled mainly by dissociative electron attachment. Above 15 eV, the portion of a yield function that increases linearly is attributed to nonresonant processes, such as dipolar dissociation. A resonant structure is superimposed on this signal around 20 eV in the anion yield functions. This structure implicates dissociative electron attachment and/or resonant decay of the transient anion into the dipolar dissociation channel, with a minimal contribution from multiple inelastic electron scattering. The yields of all desorbing anions clearly show that electron resonances contribute to the damage of all DNA bases bombarded with 5-40 eV electrons. Comparison of the ion yields indicates that adenine is the least sensitive base to slow electron attack. Electron-irradiated guanine films exhibit the largest yields of desorbed anions.     

The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors.

It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- 2.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 +/- 1.8 and 20.5 +/- 3.2 microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.2 +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.2 microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.     

Accumulation of N-Arachidonoylethanolamine (Anandamide) into Cerebellar Granule Cells Occurs via Facilitated Diffusion

Abstract: N -Arachidonoylethanolamine (anandamide, AEA) is a putative endogenous ligand of the cannabinoid receptor. Intact cerebellar granule neurons in primary culture rapidly accumulate AEA. [³H]AEA accumulation by cerebellar granule cells is dependent on incubation time ( t _1/2 of 2.6 ± 0.8 min at 37°C) and temperature. The accumulation of AEA is saturable and has an apparent K _m of 41 ± 15 µ M and a V _max of 0.61 ± 0.04 nmol/min/10⁶ cells. [³H]AEA accumulation by cerebellar granule cells is significantly reduced by 200 µ M phloretin (57.4 ± 4% of control) in a noncompetitive manner. [³H]AEA accumulation is not inhibited by either ouabain or removal of extracellular sodium. [³H]AEA accumulation is fairly selective for AEA among other naturally occurring N -acylethanolamines; only N -oleoylethanolamine significantly inhibited [³H]AEA accumulation at a concentration of 10 µ M . The ethanolamides of palmitic acid and linolenic acid were inactive at 10 µ M . N -Arachidonoylbenzylamine and N -arachidonoylpropylamine, but not arachidonic acid, 15-hydroxy-AEA, or 12-hydroxy-AEA, compete for AEA accumulation. When cells are preloaded with [³H]AEA, temperature-dependent efflux occurs with a half-life of 1.9 ± 1.0 min. Phloretin does not inhibit [³H]AEA efflux from cells. These results suggest that AEA is accumulated by cerebellar granule cells by a protein-mediated transport process that has the characteristics of facilitated diffusion.     

Base tilt of B-form poly[d(G)]-poly[d(C)] and the B- and Z-conformations of poly[d(GC)]-poly[d(GC)] in solution

We have measured the CD, isotropic absorption, and linear dichroism (LD) in the vacuum-uv spectral region for the B-conformations of poly[d(G)]-poly[d(C)] and poly[d(GC)]-poly[d(GC)], and for the Z-conformation of poly[d(GC)]-poly[d(GC)] formed in 70% trifluoroethanol. The reduced dichroism (LD divided by isotropic absorption) for all conformations varied with wavelength, indicating that the bases are not perpendicular to the helix axis. Since the directions of the transition dipoles are known, the inclinations and axes of inclination of each base can be determined from the wavelength dependence of the reduced dichroism spectra. The results indicate that the base normals of the (G + C) polymers in the B- and Z-conformations are tilted at angles greater than 19° with respect to the helix axis. The guanine and cytosine bases have different inclinations, and the tilt axes are not parallel. Therefore, the bases for all the (G + C) polymer conformations studied are buckled and propeller twisted.     

Adenosyl coenzyme and pH dependence of the [4Fe-4S]2+/1+ transition in lysine 2,3-aminomutase

5'-[N-[(3S)-3-Amino-carboxypropyl]-N-methylamino]-5(')-deoxyadenosine (azaSAM), an analog of S-adenosyl-L-methionine (SAM), was used to study the cofactor-dependent reduction of the [4Fe-4S](2+) center in lysine 2,3-aminomutase to the +1 oxidation state. azaSAM has a tertiary nitrogen in place of the sulfonium center of SAM. The analog binds to lysine 2,3-aminomutase with K(d)s of 1.4+/-0.3 microM at pH 8.0 and 2.2+/-0.6 microM at pH 6.5. Reduction of the [4Fe-4S](2+) center in the presence of this analog gives a 10K [4Fe-4S](1+) electron paramagnetic resonance (EPR) signal similar to that seen with SAM or S-adenosyl-L-homocysteine (SAH). The pH dependence of cofactor-induced reduction was examined to determine whether ionization of the tertiary nitrogen (pK(a)=7.08) might affect reduction of the [4Fe-4S](2+) center. The results show similar behavior in azaSAM and SAH, demonstrating that ionization of the aza group in azaSAM does not account for pH dependence in cofactor-dependent reduction of the [4Fe-4S](2+) center. The signal shape of the low-temperature EPR signal for the [4Fe-4S](1+) center in the SAM-induced reduction displayed a pH dependence that was not observed in the azaSAM- or SAH-induced spectra. Unique features of the signal are at a maximum at the pH activity optimum of pH 8 and are diminished as the pH is lowered or raised. These features are also absent in the spectra at all pHs examined when reduction is induced by azaSAM or SAH.     

A rearranging ligand enables allosteric control of catalytic activity in copper-containing nitrite reductase

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A(460)/A(600)=0.71) differs significantly from that of the native nitrite reductase (A(460)/A(600)=1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E(M)=312(+/-5)mV versus hydrogen) is higher than that of the native enzyme (E(M)=213(+/-5)mV). M150G has a lower catalytic activity (k(cat)=133(+/-6)s(-1)) than the wild-type nitrite reductase (k(cat)=416(+/-10)s(-1)). The binding of external ligands to M150G restores spectral properties, midpoint potential (E(M)<225mV), and catalytic activity (k(cat)=374(+/-28)s(-1)). Also the M150H (A(460)/A(600)=7.7, E(M)=104(+/-5)mV, k(cat)=0.099(+/-0.006)s(-1)) and M150T (A(460)/A(600)=0.085, E(M)=340(+/-5)mV, k(cat)=126(+/-2)s(-1)) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.     

Waterpipe Tobacco Smoking and Gastric Cancer Risk among Vietnamese Men

BackgroundThe association of waterpipe tobacco (WPT) smoking with gastric cancer (GC) risk was suggested.MethodsA hospital-based case-control study was conducted to examine the association of WPT with GC risk among Vietnamese men, in Hanoi city, during the period of 2003–2011. Newly-diagnosed GC cases (n = 454) and control patients (n = 628) were matched by age (+/- 5 years) and the year of hospitalization. Information on smoking and alcohol drinking habits and diet including salty food intake and fruits/vegetables consumption were obtained by the interview. Maximum likelihood estimates of odds ratios (ORs) and corresponding 95% confidence intervals (Cis) were obtained using conditional logistic regression models.ResultsThe group with the highest consumption of citrus fruits showed a significantly low GC risk (OR = 0.6, 95%CI = 0.4–0.8, P for trend = 0.002). However, there was no association of raw vegetable consumption with GC risk. Referring to never smokers, GC risk was significantly higher in current WPT smokers (OR = 1.8, 95%CI = 1.3–2.4), and it was more evident in exclusively WPT smokers (OR = 2.7, 95%CI = 1.2–6.5). GC risk tended to be higher with daily frequency and longer duration of WPT smoking but these trends were not statistically significant (P for trend: 0.144 and 0.154, respectively). GC risk of those who started smoking WPT before the age of 25 was also significantly high (OR = 3.7, 95%CI = 1.2–11.3). Neither cigarette smoking nor alcohol drinking was related to GC risk.ConclusionThe present findings revealed that WPT smoking was positively associated with GC risk in Vietnamese men.     

Identification of FX in the heliobacterial reaction center as a [4Fe-4S] cluster with an S = 3/2 ground spin state

Type I homodimeric reaction centers, particularly the class present in heliobacteria, are not well understood. Even though the primary amino acid sequence of PshA in Heliobacillus mobilis has been shown to contain an F(X) binding site, a functional Fe-S cluster has not been detected by EPR spectroscopy. Recently, we reported that PshB, which contains F(A)- and F(B)-like Fe-S clusters, could be removed from the Heliobacterium modesticaldum reaction center (HbRC), resulting in 15 ms lifetime charge recombination between P798(+) and an unidentified electron acceptor [Heinnickel, M., Shen, G., Agalarov, R., and Golbeck, J. H. (2005) Biochemistry 44, 9950-9960]. We report here that when a HbRC core is incubated with sodium dithionite in the presence of light, the 15 ms charge recombination is replaced with a kinetic transient in the sub-microsecond time domain, consistent with the reduction of this electron acceptor. Concomitantly, a broad and intense EPR signal arises around g = 5 along with a minor set of resonances around g = 2 similar to the spectrum of the [4Fe-4S](+) cluster in the Fe protein of Azotobacter vinelandii nitrogenase, which exists in two conformations having S = (3)/(2) and S = (1)/(2) ground spin states. The M?ssbauer spectrum in the as-isolated HbRC core shows that all of the Fe is present in the form of a [4Fe-4S](2+) cluster. After reduction with sodium dithionite in the presence of light, approximately 65% of the Fe appears in the form of a [4Fe-4S](+) cluster; the remainder is in the [4Fe-4S](2+) state. Analysis of the non-heme iron content of HbRC cores indicates an antenna size of 21.6 +/- 1.1 BChl g molecules/P798. The evidence indicates that the HbRC contains a [4Fe-4S] cluster identified as F(X) that is coordinated between the PshA homodimer; in contrast to F(X) in other type I reaction centers, this [4Fe-4S] cluster exhibits an S = (3)/(2) ground spin state.     

New synthesis and evaluation of enantiomers of 7-methyl-2-exo-(3'-iodo-5'-pyridinyl)-7-azabicyclo[2.2.1]heptane as stereoselective ligands for PET imaging of nicotinic acetylcholine receptors

A simple and efficient synthesis of nAChR antagonist (+/-)-7-methyl-2-exo-(3'-iodo-5'-pyridinyl)-7-azabicyclo[2.2.1]-heptane ((+/-)-NMI-EPB) has been developed. Both enantiomers of (+/-)-NMI-EPB were separated by semi-preparative chiral HPLC. The enantiomers manifested a substantial difference in their inhibition binding affinities ((+)-NMI-EPB, K(i)=2310, 1680 pM; (-)-NMI-EPB, K(i)=55, 68 pM). The enantiomers were stereoselectively radiolabeled with (11)C. In the distribution studies in the rodent brain [(11)C](-)-NMI-EPB specifically labeled nAChR whereas [(11)C](+)-NMI-EPB exhibited little specific binding. In the baboon PET study [(11)C](-)-NMI-EPB did not reach steady-state within 90 min post-injection suggesting that the radioligand may have some limitations for quantitative imaging.     

Stretch induced accumulation of total Ca and Na in cytosol and nucleus: a comparison between cardiac trabeculae and isolated myocytes

By means of electron probe microanalysis (EPMA), we quantified changes in total sodium [Na] and calcium [Ca] concentration owing to the following: (i) local axial stretch (LAS) of isolated rat myocytes and (ii) end-to-end stretch (ETES) of rat ventricular trabeculae. For LAS, the distance between patch pipette and a cell-attached stylus was increased by maximally 20%; this activated a nonselective cationic current I(SAC) of approximately -0.5 nA, which was blocked by streptomycin. Trabeculae were stretched end-to-end from 85% L(max) to L(max). Stretch increased cytosolic [Na](total) by 34% in isolated myocytes (p < 0.001) and by 43% in trabeculae (p < 0.001). The increment in nuclear [Na](total) was 21% in myocytes (p < 0.01) and 20% in trabeculae (p < 0.001). Stretch increased [Ca](total) in isolated myocytes, in both cytosol (from 0.63 +/- 0.09 to 1.09 +/- 0.20 mmol/L, p < 0.05) and nucleus (from 0.33 +/- 0.05 to 0.64 +/- 0.13 mmol/L, p < 0.05). In trabeculae, the stretch-induced increment of 51% in cytosolic [Ca](total) remained nonsignificant (p < 0.15). In the nucleus, [Ca](total) did not change. We interpret the difference of stretch on nuclear calcium in myocytes vs. trabeculae with the assumption that LAS, but not ETES, produces shear-stress components that translate the mechanical stimulus deeply into the cell where it may modulate [Ca](total) by signals independent of I(SAC).     

Levels, metabolism, and pharmacological activity of anandamide in CB(1) cannabinoid receptor knockout mice: evidence for non-CB(1), non-CB(2) receptor-mediated actions of anandamide in mouse brain

Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB(1)), yet some of the neurobehavioral effects of this compound in mice are unaffected by a selective CB(1) antagonist. We studied the levels, pharmacological actions, and degradation of AEA in transgenic mice lacking the CB(1) gene. We quantified AEA and the other endocannabinoid, 2-arachidonoyl glycerol, in six brain regions and the spinal cord by isotope-dilution liquid chromatography-mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB(1) distribution only in part. In CB(1) knockout homozygotes (CB(1)-/-), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild-type (CB(1)+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB(1) in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2-Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB(1)-/- with respect to CB(1)+/+ mice in all regions. AEA and Delta(9)-tetrahydrocannabinol (THC) were tested in CB(1)-/- mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB(1)-/- mice. AEA, but not THC, stimulated GTPgammaS binding in brain membranes from CB(1)-/- mice, and this stimulation was insensitive to CB(1) and CB(2) antagonists. We suggest that non-CB(1), non-CB(2) G protein-coupled receptors might mediate in mice some of the neuro-behavioral actions of AEA.     

Electron attachment to DNA single strands: gas phase and aqueous solution

The 2′-deoxyguanosine-3′,5′-diphosphate, 2′-deoxyadenosine-3′,5′-diphosphate, 2′-deoxycytidine-3′,5′-diphosphate and 2′-deoxythymidine-3′,5′-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3′,5′-dTDP (0.17 eV) and 3′,5′-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3′,5′-dTDP > 3′,5′-dCDP > 3′,5′-dGDP > 3′,5′-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3′-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3′-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3′,5′-dADP⁻ (0.26 eV) and 3′,5′-dGDP⁻ (0.32 eV) indicate that electron detachment might compete with reactions having high activation barriers such as glycosidic bond breakage. However, the radical anions of the pyrimidine nucleotides with high VDE are expected to be electronically stable. Thus the base-centered radical anions of the pyrimidine nucleotides might be the possible intermediates for DNA single-strand breakage.     

A study on the nickel II-famotidine complexes

Potentiometric studies have shown that Ni(II) forms three pH-dependent complexes with famotidine (L), namely: [NiHL](3+), [NiL](2+) and [NiH(-2)L]. Two of them have been isolated from solution with a Ni/famotidine ratio of 1:1. At pH 6.0, a paramagnetic complex [NiL](2+) with octahedral geometry is formed in which, most likely thiazole N(9) and guanidine N(3) nitrogens are involved in the metal binding. Additionally, two water molecules and two perchlorate anions, ClO(4)(-), fulfil the coordination sphere. The second complex, [NiH(-2)L], that precipitates at pH 8 is diamagnetic and takes square-planar geometry in which four nitrogen donors: N(3), N(9), N(16) and N(20) coordinate to Ni(II). Potentiometric studies, mass spectrometry, FT-IR and Raman spectroscopy are employed to determine and discuss the structure of both complexes. Additionally, 1H, 13C and 15N NMR spectroscopy is used to confirm the binding site in a square-planar complex. The assignment of vibrational bands are made using ab initio HF/CEP-31G method.     

Electrochemical oxidation of purine and pyrimidine bases based on the boron-doped nanotubes modified electrode

Based on the excellent physicochemical properties of boron-doped carbon nanotubes (BCNTs), the electrochemical analysis of four free DNA bases at the BCNTs modified glassy carbon (GC) electrode was investigated. Herein, the BCNTs/GC electrode exhibited remarkable electrocatalytic activity towards the oxidation of purine bases (guanine (G), adenine (A)). More significantly, the direct oxidation of pyrimidine bases (thymine (T), cytosine (C)) was realized. It may be due to that BCNTs have the advantages of high electron transfer kinetics, large surface area, prominent antifouling ability and electrode activity. On basis of this, a novel and simple strategy for the determination of G, A, T and C was proposed. The BCNTs/GC electrode showed high sensitivity, wide linear range and capability of detection for the electrochemical determination of G, A, T, and C. On the other hand, the electrochemical oxidation of quaternary mixture of G, A, T, and C at the BCNTs/GC electrode was investigated. It was obtained that the peak separation between G and A, A and T, T and C were large enough for their potential recognition in mixture without any separation or pretreatment. The BCNTs/GC electrode also displayed good stability, reproducibility and excellent anti-interferent ability. Therefore, it can be believed that the BCNTs/GC electrode would provide a potential application for the electrochemical detection of DNA in the field of genetic-disease diagnosis.