Donepezil-tacrine hybrid related derivatives as new dual binding site inhibitors of AChE

A new series of donepezil–tacrine hybrid related derivatives have been synthesised as dual acetylcholinesterase inhibitors that could bind simultaneously to the peripheral and catalytic sites of the enzyme. These new hybrids combined a tacrine, 6-chlorotacrine or acridine unit as catalytic binding site and indanone (the heterocycle present in donepezil) or phthalimide moiety as peripheral binding site of the enzyme, connected through a different linker tether length. One of the synthesised compounds emerged as a potent and selective AChE inhibitor, which is able to displace propidium in a competition assay. These results seem to confirm the ability of this inhibitor to bind simultaneously to both sites of the enzyme and make it a promising lead for developing disease-modifying drugs for the future treatment of Alzheimer’s disease. To gain insight into the molecular determinants that modulate the inhibitory activity of these compounds, a molecular modelling study was performed to explore their binding to the enzyme.     

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.     

Differential crop damage by healthy and nucleopolyhedrovirus-infected Mamestra brassicae L. (Lepidoptera: Noctuidae) larvae: a field examination

Baculovirus infection in Lepidoptera can alter both larval mobility and feeding rates, which can in turn affect pathogen transmission and dispersal in the field. We compared the damage to cabbage plants in the field caused by healthy and nucleopolyhedrovirus-infected Mamestra brassicae L. (Lepidoptera: Noctuidae) larvae released as second and fourth instars. There was no significant difference in plant consumption by healthy and infected larvae for the first 4 days after release. From day 5 onwards, infected larvae caused significantly less defoliation. This pattern was similar for larvae at both larval instars. Defoliation was greater for fourth instars throughout the experiment.     

Use of the paired samples (cerebrospinal fluid and serum) in immunodiagnostic of active and inactive human neurocysticercosis

Paired samples of cerebrospinal fluid (CSF) and serum of 30 patients--10 with active, 10 with inactive neurocysticercosis (NCC), and 10 control subjects--were evaluated by enzyme-linked immunosorbent assay (ELISA) using two Taenia crassiceps metacestode extracts as antigen in order to detect IgG antibodies. In active NCC, high levels of IgG were detected (p < 0.05). The CSF samples showed 80% (CI 72-88) of reactivity in the saline extract (S) and 90% (CI 84-95) in sodium dodecyl sulphate (SDS) and the serum samples were reactive in 90% (CI 84-95) and 100% (CI 98-100) in the S and SDS antigenic extracts, respectively. The use of the paired samples of CSF and serum in active NCC showed equivalent results suggesting that the serum samples could be used as a screening in those patients whose CSF puncture is counter-indicated.     

Exploring the microbial biodegradation and biotransformation gene pool

Similar to the New World explorers of the 16th and 17th century, microbiologists today find themselves at the edge of unknown territory. It is estimated that only 0.1-1% of microorganisms can be cultivated using current techniques; the vastness of microbial lifestyles remains to be explored. Because the microbial metagenome is the largest reservoir of genes that determine enzymatic reactions, new techniques are being developed to identify the genes that underlie many valuable chemical biotransformations carried out by microbes, particularly in pathways for biodegradation of recalcitrant and xenobiotic molecules. Our knowledge of catabolic routes built on research during the past 40 years is a solid basis from which to venture on to the little-explored pathways that might exist in nature. However, it is clear that the vastness of information to be obtained requires astute experimental strategies for finding novel reactions.     

Synergistic inhibition of respiration in brain mitochondria by nitric oxide and dihydroxyphenylacetic acid (DOPAC). Implications for Parkinson's disease

The inhibition of mitochondrial respiration by nitric oxide (.NO) at cytochrome c oxidase level has been established as a physiological regulatory mechanism of mitochondrial function. Given, on the one hand, the potential involvement of .NO and dopamine metabolism in mitochondrial dysfunction associated with neurodegeneration and, on the other hand, the reported interaction of .NO with dihydroxyphenylacetic acid (DOPAC), a major mitochondrial-associated dopamine metabolite, we examined the combined effects of .NO and DOPAC on the respiratory chain of isolated rat brain mitochondria. Whereas dopamine or DOPAC induced no measurable effects on the mitochondrial respiration rate, a mixture of .NO with DOPAC inhibited the rate in a way stronger than that exerted by .NO. This effect was noticed with actively respiring (state 3) and resting (state 4) mitochondria. At variance with DOPAC, dopamine failed to potentiate .NO inhibitory effects. The inhibition was dependent on the concentration of both compounds, .NO and DOPAC, and exhibited characteristics similar to those exerted by .NO, namely: it was reversible and dependent on the concentration of oxygen. Analysis of respiratory enzymatic activities demonstrated a selective inhibition at the level of cytochrome c oxidase (complex IV). Insights into the chemical mechanisms underlying the inhibitory effect were inferred from experiments using metmyoglobin (a ligand for .NO and derived species, such as nitroxyl anion) and ferrocyanide (a reductant of .NO, producing nitroxyl anion). Whereas metmyoglobin decreased the inhibition, ferrocyanide potentiated the inhibition. Moreover, a mixture of ferrocyanide with .NO reproduced the effects exerted by the mixture of .NO with DOPAC. The results are consistent with the notion of a reaction of .NO with DOPAC producing a nitric oxide-derived compound(s), which inhibit O2 uptake at the cytochrome oxidase level. Although the mechanism in question remains to be clearly elucidated it is suggested that the .NO/DOPAC-dependent inhibition of cytochrome oxidase may involve nitroxyl anion. The significance of these observations for mitochondrial dysfunction inherent in Parkinson's disease is discussed.     

Environmental effects on the fluorescence behaviour of carbazole derivatization reagents.

The carbazole ring is the basic structure present in the fluorescence derivatization reagents 9-chlorocarbonylcarbazole and 9-carbazolylacetic acid. The fluorescence behaviour of these carbazole derivatives was studied in solvents with different polarities (cyclohexane, ethanol, acetonitrile, water) and at different pH values (4.5 and 8.8). The influence of the low polarity environment afforded by 2-hydroxypropyl-beta-cyclodextrin (HPbeta-CD) is also described. The behaviour of the fluorescent reagents is compared to the model molecules carbazole and 9-methylcarbazole. For all derivatives studied, a bathochromic shift in the fluorescence emission maxima was observed when the solvent polarity was increased. A bathochromic shift was observed in dioxane solutions, which can be ascribed to the peculiar behaviour of this solvent. The changes in the fluorescence intensity in the case of 9-carbazolylacetic acid can be related to the ionization of the carboxylic acid group. Inclusion into the cavity of HPbeta-CD allows emission spectra to be obtained close to those obtained in ethanolic solutions with a remarkable enhancement in the fluorescence intensity, depending on the chemical structure of the carbazole derivative included.     

Diversity of non-reducing polyketide synthase genes in the Pertusariales (lichenized Ascomycota): a phylogenetic perspective

Lichenized fungi synthesize a great variety of secondary metabolites. These are typically crystalline compounds, which are deposited extracellularly on the fungal hyphae. While we know a lot about the chemical properties and structures of these substances, we have very little information on the molecular background of their biosynthesis. In the current study we analyze the diversity of non-reducing polyketide synthase (PKS) genes in members of the lichenized Pertusariales. This order primarily contains fully oxidized secondary metabolites from different substance classes, and is chemically and phylogenetically well studied. Using a degenerate primer approach with subsequent cloning we detected up to five non-reducing PKS sequences in a single PCR product. Eighty-five new KS sequence fragments were obtained for this study. Analysis of the 157 currently available fungal KS sequence fragments in a Bayesian phylogenetic framework revealed 18 highly supported clades that included only lichenized taxa, only non-lichenized taxa, or both. Some Pertusarialean groupings of PKS sequences corresponded partly to phylogenetic groupings based on ribosomal DNA. This is reasonable, because a correlation between well-supported phylogenetic lineages and the occurrence of secondary metabolites in the Pertusariales has been observed before. However, no clear linkage was found between the PKS genes analyzed and the ability to produce a particular secondary substance. Several PKS clades did not reveal obvious patterns of secondary compound distribution or phylogenetic association. Compared with earlier phylogenetic analyses of KS sequences the increased sampling in the current study allowed us to detect many new groupings within the fungal non-reducing PKSs.