Active site mutant acetylcholinesterase interactions with 2-PAM, HI-6, and DDVP

We used mouse recombinant wild-type acetylcholinesterase (AChE; EC 3.1.1.7), butyrylcholinesterase (BChE; EC 3.1.1.8), and AChE mutants with mutations (Y337A, F295L, F297I, Y72N, Y124Q, and W286A) that resemble residues found at structurally equivalent positions in BChE, to find the basis for divergence between AChE and BChE in following reactions: reversible inhibition by two oximes, progressive inhibition by the organophosphorus compound DDVP, and oxime-assisted reactivation of the phosphorylated enzymes. The inhibition enzyme-oxime dissociation constants of AChE w.t. were 150 and 46 microM, of BChE 340 and 27 microM for 2-PAM and HI-6, respectively. Introduced mutations lowered oxime binding affinities for both oximes. DDVP progressively inhibited cholinesterases yielding symmetrical dimethylphosphorylated enzyme conjugates at rates between 104 and 105/min/M. A high extent of oxime-assisted reactivation of all conjugates was achieved, but rates by both oximes were up to 10 times slower for phosphorylated mutants than for AChE w.t.     

Dietary habits and folate status in women of childbearing age in Croatia

Folic acid and folates have an important role in prevention of neural tube defect that appears in the first weeks of pregnancy, when women are still not aware of their pregnancy, especially when pregnancy is not planned. Since ensuring sufficient quantities of folates and folic acid in this period is essential, dietary habits of childbearing age women are very important. In line with that the intake of folates and folic acid in nutrition of women age group 20-30 years is examined, as well as the frequency of consumption of foodstuffs rich in vitamins and folic acid supplements. Values of folates in serum are presented, dependent on their nutritional habits. Obtained results indicate that in spite of inadequate intake of folates and folic acid from foodstuffs, clinical deficit is not recorded, which is the result of frequent consumption of dietary supplements. In accordance with these dietary habits, differences in the folates status of examinees were observed.     

Amino acid residues involved in stereoselective inhibition of cholinesterases with bambuterol

Bambuterol is a chiral carbamate known as selective inhibitor of butyrylcholinesterase (BChE). In order to relate bambuterol selectivity and stereoselectivity of cholinesterases to the active site residues, we studied the inhibition of recombinant mouse BChE, acetylcholinesterase (AChE) and six AChE mutants, employed to mimic BChE active site residues, by bambuterol enantiomers. Both enantiomers selectively inhibited BChE about 8000 times faster than AChE. The largest inhibition rate increase in comparison to AChE w.t. was observed with the F295L/Y337A mutant, showing that leucine 295 and alanine 337 are crucial residues in BChE for high bambuterol selectivity. All studied enzymes preferred inhibition by the R- over the S-bambuterol. The enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations (Y337A, F295L/Y337A and F297I/Y337A) increased, in comparison to w.t. enzymes, inhibition rate constants of R- bambuterol more than that of S- bambuterol resulting in four times higher stereoselectivity. Peripheral site mutations (Y124Q and Y72N/Y124Q/Y337A) increased inhibition rate by S- more than R-bambuterol and consequently diminished the stereoselectivity.     

Interactions of butane, but-2-ene or xylene-like linked bispyridinium para-aldoximes with native and tabun-inhibited human cholinesterases

Kinetic parameters were evaluated for inhibition of native and reactivation of tabun-inhibited human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7) and human plasma butyrylcholinesterase (BChE, EC 3.1.1.8) by three bispyridinium para-aldoximes with butane (K074), but-2-ene (K075) or xylene-like linker (K114). Tested aldoximes reversibly inhibited both cholinesterases with the preference for binding to the native AChE. Both cholinesterases showed the highest affinity for K114 (K(i) was 0.01mM for AChE and 0.06mM for BChE). The reactivation of tabun-inhibited AChE was efficient by K074 and K075. Their overall reactivation rate constants were around 2000min(-1)M(-1), which is seven times higher than for the classical bispyridinium para-aldoxime TMB-4. The reactivation of tabun-inhibited AChE assisted by K114 was slow and reached 90% after 20h. Since the aldoxime binding affinity of tabun-inhibited AChE was similar for all tested aldoximes (and corresponded to their K(i)), the rate of the nucleophilic displacement of the phosphoryl-moiety from the active site serine was the limiting factor for AChE reactivation. On the other hand, none of the aldoximes displayed a significant reactivation of tabun-inhibited BChE. Even after 20h, the reactivation maximum was 60% for 1mM K074 and K075, and only 20% for 1mM K114. However, lower BChE affinities for K074 and K075 compared to AChE suggest that the fast tabun-inhibited AChE reactivation by these compounds would not be obstructed by their interactions with BChE in vivo.     

Synthesis and biological evaluation of O-methyl and O-ethyl NSAID hydroxamic acids

This paper reports the synthesis of O-methyl and O-ethyl NSAID hydroxamic acids, their antimicrobial activities, and their ability to inhibit urease and soybean lipoxygenase activities. Ibuprofen and fenoprofen hydroxamic acids with free hydroxy groups present the highest antimicrobial activity, while indomethacin and diclofenac analogs show significantly lower antimicrobial activity. Diclofenac hydroxamic acid 4e exerts the highest anti-urease activity. Indomethacin O-ethyl hydroxamic acid 3h and ibuprofen O-benzyl hydroxamic acid 4b exert significant inhibitory activities on soybean lipoxygenase. Fenoprofen and indomethacin O-ethyl hydroxamic acids 3b and 3h and diclofenac and indomethacin O-benzyl analogs 4g and 4i highly inhibit lipid peroxidation. The highest antioxidant activity was shown by fenoprofen derivative 3b.     

1-H MRS changes in dorsolateral prefrontal cortex after donepezil treatment in patients with mild to moderate Alzheimer's disease

Magnetic resonance spectroscopy (MRS) noninvasively provides information on the concentration of some cerebral metabolites in vivo. Among those measurable by proton magnetic resonance spectroscopy (1H-MRS), N-acetyl-aspartate (NAA) is decreased, and myo-inositol (ml) and choline (Cho) levels are increased in patients with Alzheimer's disease (AD). Donepezil, an acetylcholinesteraze inhibitor, has proven effect on cognitive symptoms in patients with AD. In previous studies, treatment response was associated with an increase of NAA and NAA/Cr in the parietal lobe and hippocampi. Correlation of longitudinal changes of 1H-MRS detectable metabolites in dorsolateral prefrontal cortex (DLPFC) with clinically observable changes is a poorly researched topic. The objective of this non-interventional study is to assess whether changes in 1H-MRS measurable metabolites correlate with clinical outcome after donepezil treatment. Twelve patients with mild to moderate AD were evaluated during 26 weeks of donepezil treatment. 1H-MRS parameters in DLPFC were assessed before and after 26 weeks of donepezil treatment. Cognition was assessed with Alzheimer's Disease Assessment Scale cognitive subscale (ADAS-Cog). A significant increase in NAA/Cr ratio and significantly lower decrease in mI/Cr ratio were found in AD patients with positive treatment response. The results of this study indicate possible modest donepezil effect on prevention of neuronal functional deterioration in DLPFC which correlates with clinical outcome and point the use of 1HMRS as technique of help in assessment of drug effect.     

Ecology and taxonomy of potentially toxic Pseudo-nitzschia species in Lim Bay (north-eastern Adriatic Sea)

The population dynamics of Pseudo-nitzschia in relation to environmental factors was investigated from March 2002 to July 2008 in Lim Bay, in the north-eastern Adriatic Sea. Domoic acid was monitored in the breeding population of Mytilus galloprovincialis from 2005 to 2008. The principal-component analysis of environmental parameters showed that the system is mostly temperature driven. The phytoplankton community was mainly composed of diatoms. Pseudo-nitzschia was the dominant diatom, present in 60% of samples, with a maximum (1.6 × 10⁶ cells L⁻¹) contribution up to 97% of the total diatom abundance. Morphological analysis revealed Pseudo-nitzschia manii and potentially toxic Pseudo-nitzschia pseudodelicatissima, Pseudo-nitzschia pungens, Pseudo-nitzschia fraudulenta and Pseudo-nitzschia calliantha as the dominant species in blooms. Pseudo-nitzschia abundance positively correlated to temperature, phosphate and ammonia in accordance with its maximal abundance in the summer/autumn period when fish farms had a maximum impact on the environment. Domoic acid was detected in M. galloprovincialis in concentrations below regulatory limits, ranging from 0.097 to 0.8721 μg g⁻¹ in five cases from April to October 2005 in Lim Bay, but so far it is not clear which of the species was responsible for DA production. This study is also the first record of P. manii, P pungens and P. fraudulenta species in the Adriatic Sea.     

Increase in specific density of levobupivacaine and fentanyl solution ensures lower incidence of inadequate block

The clinical presentation of a subarachnoid block (SAB) is dependent upon the intrathecal spread of local anesthetic (LA). Intrathecal distribution depends on the chemical and physical characteristics of LA, puncture site, technique used, patient anatomical characteristics and hydrodynamic properties of cerebrospinal fluid. We tried to determine whether a combined glucose/LA solution can render a clinically significant difference in sensory block distribution and motor block intensity.This was a controlled, randomized and double blinded study. The surgical procedures were stripping of the great or small saphenous vein and extirpation of remaining varicose veins. The study included 110 patients distributed into two groups: Hyperbaric (7.5 mg levobupivacaine (1.5 ml 0.5% Chirocaine) + 50 microg Fentanyl (0.5 ml Fentanil) and 1 ml 10% glucose (Pliva)) vs. Hypobaric (7.5 mg levobupivacaine (1.5 ml 0.5% Chirocaine) + 50 microg Fentanyl (0.5 ml Fentanil) and 1 ml 0.9% NaCl (Pliva, Zagreb)) adding to a total volume of 3.5 ml per solution. Spinal puncture was at L3-L4 level. Spinal block distribution was assessed in five minute intervals and intensity of motor block was assessed according to the modified Bromage scale. Pain was assessed with the Visual Analogue Scale. A statistically significant difference in sensory block distribution, motor block intensity and recovery time was established between hyperbaric and hypobaric solutions. By increasing the specific density of anesthetic solution, a higher sensory block, with lesser variability, a diminished influence of Body Mass Index, decreased motor block intensity and faster recovery time may be achieved.