Phytotoxicity of hydrogen fluoride and fluoroborate and their uptake from solution culture by Lycopersicon esculentum and Avena sativa

The aims of this paper were to determine the phytoavailability and phytotoxicity of hydrogen fluoride (HF) and fluoroborate (BF ₄ ^- ) in solution when exposed to the root of the plant. As fluoroborate undergoes a slow hydrolysis to F and borate ions, the stability of BF ₄ ^- under solution culture conditions was determined. Fluoroborate was found to have a zero order rate constant of 0.0136 and took approximately 72 days to hydrolyse completely.Tomato (Lycopersicon esculentum) and oat (Avena sativa) plants were grown in dilute nutrient solutions which contained a range of activities of HF and BF ₄ ^- . Dry matter production of both tomato and oat plants grown in nutrient solutions were found to be restricted by increased activity of HF and BF ₄ ^- in solution. Tomatoes were more sensitive to HF and BF ₄ ^- than oats. Limitations to dry matter production coincided with increased uptake of F for F concentrations in tissue of both tomatoes and oats. Fluoride uptake of both HF and BF by tomatoes and oats was orders of magnitude higher compared to similar activities of other ionic species of F reported in previous studies. Possible mechanisms of uptake are discussed.     

High-performance liquid chromatography determination of ketone bodies in human plasma by precolumn derivatization with p-nitrobenzene diazonium fluoroborate

We developed and validated a sensitive and convenient high-performance liquid chromatography (HPLC) method for the specific determination of ketone bodies (acetoacetate and d-3-hydroxybutyrate) in human plasma. p-Nitrobenzene diazonium fluoroborate (diazo reagent) was used as a precolumn derivatization agent, and 3-(2-hydroxyphenyl) propionic acid was used as an internal standard. After the reaction, excess diazo reagent and plasma proteins were removed by passing through a solid-phase cartridge (C₁₈). The derivatives retained on the cartridge were eluted with methanol, introduced into the HPLC system, and then detected with UV at 380 nm. A calibration curve for acetoacetate standard solution with a 20-μl injection volume showed good linearity in the range of 1 to 400 μM with a 0.9997 correlation coefficient. For the determination of d-3-hydroxybutyrate, it was converted to acetoacetate before reaction with the diazo reagent by an enzymatic coupling method using d-3-hydroxybutyrate dehydrogenase and lactate dehydrogenase. A calibration curve for d-3-hydroxybutyrate standard solution also showed good linearity in the range of 1.5 to 2000 μM with a 0.9988 correlation coefficient. Analytical recoveries of acetoacetate and d-3-hydroxybutyrate in human plasma were satisfactory. The method was successfully applied to samples from diabetic patients, and results were consistent with those obtained using the thio-NAD enzymatic cycling method used in clinical laboratories.     

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent.In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i