Atomic interactions of neonicotinoid agonists with AChBP: molecular recognition of the distinctive electronegative pharmacophore

Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 A in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids.     

Evaluation of genome damage and its relation to oxidative stress induced by glyphosate in human lymphocytes in vitro

In the present study we evaluated the genotoxic and oxidative potential of glyphosate on human lymphocytes at concentrations likely to be encountered in residential and occupational exposure. Testing was done with and without metabolic activation (S9). Ferric‐reducing ability of plasma (FRAP), thiobarbituric acid reactive substances (TBARS) and the hOGG1 modified comet assay were used to measure glyphosate's oxidative potential and its impact on DNA. Genotoxicity was evaluated by alkaline comet and analysis of micronuclei and other nuclear instabilities applying centromere probes. The alkaline comet assay showed significantly increased tail length (20.39 μm) and intensity (2.19%) for 580 μg/ml, and increased tail intensity (1.88%) at 92.8 μg/ml, compared to control values of 18.15 μm for tail length and 1.14% for tail intensity. With S9, tail length was significantly increased for all concentrations tested: 3.5, 92.8, and 580 μg/ml. Using the hOGG1 comet assay, a significant increase in tail intensity was observed at 2.91 μg/ml with S9 and 580 μg/ml without S9. Without S9, the frequency of micronuclei, nuclear buds and nucleoplasmic bridges slightly increased at concentrations 3.5 μg/ml and higher. The presence of S9 significantly elevated the frequency of nuclear instabilities only for 580 μg/ml. FRAP values slightly increased only at 580 μg/ml regardless of metabolic activation, while TBARS values increased significantly. Since for any of the assays applied, no clear dose‐dependent effect was observed, it indicates that glyphosate in concentrations relevant to human exposure do not pose significant health risk. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Genes coding evolutionary novel anti-carbohydrate antibodies: studies on anti-Gal production in alpha 1,3galactosyltransferase knock out mice

This study analyzes the gene repertoire coding for antibodies to an evolutionary novel immunogenic carbohydrate antigen in mice. The alpha-gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R) is an autoantigen, abundantly expressed in wild type mice, but absent in alpha 1,3galactosyltransferase knock-out (KO) mice, where it can induce the production of the anti-Gal antibody. Hybridoma clones secreting anti-Gal were isolated from different mice and their immunoglobulin genes were analyzed. All anti-Gal clones were found to be encoded by the heavy chain gene VH22.1 and light chain gene VK5.1. Moreover, one 'forbidden' anti-Gal clone, produced in a wild type mouse, was also encoded by VH 22.1 and VK 5.1. The genes coding for the different anti-Gal clones were found to contain somatic mutations and different CDR3 domains. These data imply that a highly restricted gene usage combined with junctional diversity and somatic mutations can generate new antibodies that have not been produced in the course of the evolution of a species.     

Naturally occurring variations in the human cholinesterase genes: heritability and association with cardiovascular and metabolic traits

Cholinergic neurotransmission in the central and autonomic nervous systems regulates immediate variations in and longer-term maintenance of cardiovascular function with acetylcholinesterase (AChE) activity that is critical to temporal responsiveness. Butyrylcholinesterase (BChE), largely confined to the liver and plasma, subserves metabolic functions. AChE and BChE are found in hematopoietic cells and plasma, enabling one to correlate enzyme levels in whole blood with hereditary traits in twins. Using both twin and unrelated subjects, we found certain single nucleotide polymorphisms (SNPs) in the ACHE gene correlated with catalytic properties and general cardiovascular functions. SNP discovery from ACHE resequencing identified 19 SNPs: 7 coding SNPs (cSNPs), of which 4 are nonsynonymous, and 12 SNPs in untranslated regions, of which 3 are in a conserved sequence of an upstream intron. Both AChE and BChE activity traits in blood were heritable: AChE at 48.8 ± 6.1% and BChE at 81.4 ± 2.8%. Allelic and haplotype variations in the ACHE and BCHE genes were associated with changes in blood AChE and BChE activities. AChE activity was associated with BP status and SBP, whereas BChE activity was associated with features of the metabolic syndrome (especially body weight and BMI). Gene products from cDNAs with nonsynonymous cSNPs were expressed and purified. Protein expression of ACHE nonsynonymous variant D134H (SNP6) is impaired: this variant shows compromised stability and altered rates of organophosphate inhibition and oxime-assisted reactivation. A substantial fraction of the D134H instability could be reversed in the D134H/R136Q mutant. Hence, common genetic variations at ACHE and BCHE loci were associated with changes in corresponding enzymatic activities in blood.     

Influence of local inflammation of the peritoneal membrane on diuresis and residual renal function in patients treated with peritoneal dialysis

The aim of this study was to investigate the connection between local inflammation of the peritoneal membrane and diuresis, as well as the residual renal function (RRF) in patients treated with continuous ambulatory peritoneal dialysis (CAPD). Twenty patients treated with CAPD participated in this cross-sectional study. To determine the influence of local inflammation of the peritoneal membrane, effluent interleukin-6 (IL-6) and soluble interleukin-6 receptor (sIL-6R) levels were measured. The level of IL-6, in the group as a whole, was significantly higher in effluent (7.87 pg/mL) than in serum (1.29 pg/mL). There was a significant correlation between effluent and serum IL-6 (r = 0.608; P = 0.002). There was also a significant relationship between effluent and serum IL-6 and duration of CAPD treatment, respectively (r = 0.577; P = 0.004; r = 0.528; P = 0.008). Further, there was a significant negative correlation between effluent IL-6 and daily diuresis (r = −0.533; P = 0.008), but there was no significant correlation between effluent IL-6 and RRF (r = −0.339, P = 0.072). On the other hand, the concentrations of effluent IL-6 were significantly higher in patients with RRF <2 mL/min than in those with RRF ≥2 mL/min (P = 0.039). In conclusion, local inflammation has a significant impact on the amount of diuresis and probably on RRF in patients on CAPD.     

The role of aspirin and dipyridamole on vascular DNA synthesis and intimal hyperplasia following deendothelialization

Platelets are implicated both in acute thrombotic events and, through platelet-derived growth factor, in the development of intimal hyperplasia. We have investigated, in vivo, the influence of aspirin and dipyridamole on vascular smooth muscle cell proliferation and DNA synthesis following balloon catheter injury. Fifty-eight male, New Zealand white rabbits were divided equally into two groups; the test group was fed aspirin (14 mg/kg/day) and dipyridamole (9 mg/kg/day) from 2 days prior to surgery until sacrifice at 1, 2, 3, 4, 7, 14, or 28 days after injury. All animals were sacrificed 1 h after injection of [3H]thymidine and the smooth muscle cell DNA specific activity and total kinetic activity were determined. Intimal hyperplasia was measured by light microscopy and intimal nuclear proliferation was determined by counting nuclei per millimeter of internal elastic lamina. Nuclear proliferation was maximal at 14 days (25 +/- 1.2) but intimal hyperplasia was still increasing at 28 days. DNA specific activity after 24 hr (test: 4 +/- 2 dpm/micrograms DNA; control: 3.3 +/- 3 dpm/micrograms DNA) was similar to basal levels in uninjured rabbits. DNA synthesis peaked in both groups between the second and third day (test: 177 +/- 27 dpm/micrograms DNA; control: 185 +/- 39 dpm/micrograms DNA) and then declined slowly toward baseline values. There was no significant difference between treated and normal rabbits in either [3H]thymidine incorporation, nuclear proliferation, or development of intimal hyperplasia despite 90% inhibition of platelet aggregation and a significant reduction (78%) in [14C]serotonin release following collagen challenge (6 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Editors and editing of anti-DNA receptors.

Receptor editing is a means by which immature bone marrow B cells can become self-tolerant. Rearrangements of heavy (H) and/or light (L) chain genes are induced by encounter with autoantigens to change the specificity from self to nonself. We have developed site-directed transgenic mice (sd-tg) whose transgenes code for the H chain of antibodies that bind DNA. B cells that express the transgenic H chain associate mainly with four of the 93 functional Vkappa genes of the mouse. Numerous aspartate residues that might inhibit DNA binding by the V(H) domain distinguish these L chain Vkappa sequences, but engaging these Vkappa editors often requires multiple rearrangements. Among the edited B cells is a subset of multispecific cells that express multiple receptors. One consequence of multispecificity is partial autoreactivity; these multispecific B cells may contribute to autoimmunity.     

Increased vascular contraction and sensitivity to norepinephrine after endothelial denudation is inhibited by prazosin

The vasomotor function of rabbit aorta was examined after deendothelialization with a Fogarty balloon catheter and during the subsequent development of intimal hyperplasia. Helical strips of injured lower abdominal aortic tissue showed an increase in norepinephrine-induced contraction when compared with control strips from normal upper abdominal aorta. This increase was 235% +/- 40% of control immediately after injury and 341% +/- 51% at 28 days after injury. Standardized dose-response curves demonstrated that the injured tissue was increasingly sensitive over time to norepinephrine and that this was more marked at physiologic levels of norepinephrine. Contraction was blocked by prazosin hydrochloride but not by yohimbine or propranolol. Furthermore, a single intramuscular dose of prazosin hydrochloride 2 hours before injury significantly (p = 0.001) reduced the maximal contraction and sensitivity. These results imply an increase in vasomotor reactivity after deendothelialization mediated through the alpha 1-adrenergic receptor. These functional changes are related to the pertinent morphologic observations.