The Binding of Donepezil with External Mouth of K+-Channels of Molluscan Neurons

Earlier, we have shown a strong inhibitory effect of donepezil on K⁺-current of molluscan neurons (Solntseva et al., Comp Biochem Physiol 144, 319–326, 2007). In the present work, a possible interaction of donepezil with the external mouth of the channel was examined using, as a tool, tetraethylammonium (TEA), a classical antagonist of potassium channels. Experiments were conducted in isolated neurons of snail Helix aspersa using the two-microelectrode voltage-clamp technique. A high-threshold slow-inactivating K⁺-current involving Ca²⁺-dependent (I _C) and Ca²⁺-independent (I _K) components was recorded. The I _C was estimated at 30 mV, and I _K at 100 mV. The IC₅₀ values for blocking effect of donepezil on I _C varied from 5.0 to 8.9 μM in different cells. Corresponding values for I _K varied from 4.9 to 9.9 μM. The IC₅₀ values for blocking effect of TEA on I _C lied in the range of 200 to 910 μM, and on I _K lied in the range of 100 to 990 μM. The comparison of the effects of donepezil and TEA on the same cells revealed significant correlation between IC₅₀ values of these effects. The value of Spearman coefficient of correlation (r) was 0.77 for I _C (P < 0.05), and 0.82 for I _K (P < 0.05). In the presence of TEA, the effect of donepezil, both on I _C and I _K, appears significantly weaker than in control solution. Dose–response curves of donepezil effect both on I _C and I _K were shifted right along horizontal axis when donepezil was applied in combination with TEA. Results suggest that TEA interferes with donepezil and precludes the occupation by donepezil of its own site. We suppose that the site for donepezil is situated near the TEA site with possible overlap.     

Lipids and carotid plaque in the Northern Manhattan Study (NOMAS)

Background

Heterogeneity in B-type natriuretic peptide (BNP) levels, especially among individuals with acute heart failure with normal left ventricular ejection fraction (HFNEF), can cause confusion in interpreting results. We investigated the characteristics of cases of acute HFNEF with only modestly elevated BNP.

Methods

One hundred forty-two patients with acute or acute exacerbation of chronic HFNEF were divided into two groups by BNP level: BNP < 100 pg/ml (NB group, n = 45) and BNP ≥ 100 pg/ml (B group, n = 97). We compared clinical findings, echocardiography results, and neurohormonal factors between these two groups.

Results

In the NB group, a history of open-heart surgery (OHS) was more frequent (71% vs. 22%, p < 0.0001) and hypertension was less frequent (p = 0.0005). Left atrial diameter (LAd) was higher (p = 0.0026), while interventricular septal thickness, posterior wall thickness, relative wall thickness, left ventricular mass index were lower (p = 0.0005, p = 0.0225, p = 0.0114, p = 0.0051, respectively) in the NB group. In patients with HFNEF, a history of OHS remained an independent predictor of BNP level (< 100 pg/ml) after adjustment for hypertension, age, LAd, and interventricular septal thickness (odds ratio 3.6, p = 0.0252).

Conclusion

We found associations between acute HFNEF with less elevated BNP and a history of OHS. In a patient suspected of HFNEF, a history of OHS is considered diagnostic evidence of presence of diastolic heart failure when plasma levels of BNP are less elevated.     

Organs of hearing and equilibrium during ontogenesis of mammals

The peripheral part of acoustic analyzer was studied during pre-and postnatal development of mammals. The main trends of structural evolution of the outer, middle, and inner ear were followed in representatives of different ecological groups during postnatal development. The use of ecologomorphological approach made it possible to establish that specific structural features of the hearing organ in different mammals are determined by adaptation to specific acoustic properties of the environment. It was shown that morphofunctional adaptations directed at optimization of acoustic sensitivity in different environmental conditions were leading in the hearing organ evolution. Comparative-embryological studies of the peripheral part of acoustic system made it possible to determine the stages of formation of individual structures and establish general patterns of prenatal development of the organs of hearing and equilibrium in different mammals.     

The properties and potential metabolic role of glucokinase in halotolerant obligate methanotroph <Emphasis Type="Italic">Methylomicrobium alcaliphilum</Emphasis> 20Z

Aerobic bacteria utilizing methane as the carbon and energy source do not use sugars as growth substrates but possess the gene coding for glucokinase (Glk), an enzyme converting glucose into glucose 6-phosphate. Here we demonstrate the functionality and properties of Glk from an obligate methanotroph Methylomicrobium alcaliphilum 20Z. The recombinant Glk obtained by heterologous expression in Escherichia coli was found to be close in biochemical properties to other prokaryotic Glks. The homodimeric enzyme (2 × 35 kDa) catalyzed ATP-dependent phosphorylation of glucose and glucosamine with nearly equal activity, being inhibited by ADP (K _i = 2.34 mM) but not affected by glucose 6-phosphate. Chromosomal deletion of the glk gene resulted in a loss of Glk activity and retardation of growth as well as in a decrease of intracellular glycogen content. Inactivation of the genes encoding sucrose phosphate synthase or amylosucrase, the enzymes involved in glycogen biosynthesis via sucrose as intermediate, did not prevent glycogen accumulation. In silico analysis revealed glk orthologs predominantly in methanotrophs harboring glycogen synthase genes. The data obtained suggested that Glk is implicated in the regulation of glycogen biosynthesis/degradation in an obligate methanotroph.     

Interaction of galantamine with ionic channels in molluscan neurons

Galantamine is widely used for the treatment of Alzheimer’s disease. According to the generally accepted viewpoint, its therapeutic effect is based on inhibition of acetylcholinesterase (AChE) and potentiation of nicotinic receptors. Alternative molecular targets for galanatamine, namely, voltage-gated Ca²⁺ and K⁺ channels of the neuronal membrane, are also widely discussed in the current literature. The present study is devoted to the analysis of effects of galantamine on high-threshold Ca²⁺ currents (I _Ca) and three different kinds of highthreshold K⁺ current, viz.: Ca²⁺-dependent K⁺ current (I _C), delayed rectifier (I _DR), and fast-inactivating K₊ current (I _Adepol). Experiments were conducted on molluscan neurons with the help of two-microelectrode voltageclamp technique. It was found that galantamine caused a fast, reversible and dose-dependent suppression of all types of high-threshold ionic currents. The maximal blocking effect of the alkaloid for I _Ca, I _C, and I _DR, was 100%, while for I Adepol the maximal suppression was only 60%. The mean values of IC ₅₀ for I _C, I _DR, I _Adepol, and I _Ca were 109, 237, 66, and 515 μ M, respectively, i.e., substantially higher than the corresponding values for the alkaloid-induced inhibition of AChE and potentiation of nicotinic receptors. It is concluded that the blockade of Ca₂₊ and K₊ channels has little or no contribution to the therapeutic activity of galantamine.     

Selective disturbance of associative memory reactivation by serotonin or NMDA glutamate receptor antagonists in snail

Effects of serotonin or glutamate receptors antagonists on reactivation of food aversion conditioning were studied in snail Helix lucorum. Metiotepin (nonselective serotonin receptor antagonist, 0.1 mg/snail) or MK-801 (NMDA glutamate receptor antagonist, 0.005 mg/snail) were injected 24 hours after 3 days of food aversion conditioning, then reminding stimulus (banana, "conditioned" food) was presented and food aversion conditioning was tested. Long-term impairing (more then 2 weeks) of food aversion conditioning was found 3 hours after concurrent reminding and inhibitors injection. Injection of receptor antagonists without reminding stimulus did not influence on food aversion conditioning retrieval. Besides, in snails with amnesia after metiotepin/reminder, facilitation of repeated elaboration aversion conditioning on banana is revealed. The repeated training of snails with amnesia caused by MK-801/reminder did not result in food aversion conditioning. It is was suggested that 5-HT5,6,7 serotonin receptors are involved in mechanisms of memory "trace" extraction of food aversion conditioning, whereas NMDA glutamate receptor - in processes of its storage in snail.     

Construction and Characterization of a Magnaporthe grisea Bacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of a Magnaporthe grisea bacterial artificial chromosome library. Fungal Genet. Biol. 20, 280-288. A bacterial artificial chromosome (BAC) library of Magnaporthe grisea containing 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents of M. grisea and has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using a lys1-3 auxotroph, we have shown that BAC clones at least 113 kb can be transformed into M. grisea to screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.