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.     

Bioconversion of agrowastes by Lentinula edodes: the high potential of viticulture residues

The production of four strains of edible mushroom Lentinula edodes was evaluated through solid-state fermentation (SSF) of vineyard pruning (VP), barley straw (BS), and wheat straw (WS). Biological efficiency, proximal composition, and energy value of the fruiting bodies, as well as substrate chemical changes after harvest, were determined. The shortest primordium formation time (28 days), highest biological efficiency (93.25%), highest yield (37.46%), and shortest production cycle (6 days) were observed in VP. The fruiting bodies obtained from VP had high energy value (379.09 to 392.95 kcal) and contents of protein (12.37 to 17.19%), but low contents of fat (1.82 to 2.15%). After SSF, phenol concentration decreased on VP (1.2 mmol/L) and BS (0.31 mmol/L), but on WS remained practically the same. Hemicellulose decreased in all substrates; cellulose increased on WS and decreased in the rest of the treatments. Lignin decreased on WS and BS, but its concentration increased on VP. The variability observed in the degradation capacity of lignocellulosic components was influenced by the substrate's nature, environmental factors, and genetic factors among strains. VP has great potential for shiitake production due to its low cost, short production cycles, and high biological efficiency.Research was conducted at Instituto de Ecología, AC, and Centro de Investigación en Alimentación y Desarrollo, AC     

Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His₆-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K _m values for nitrite and NADH were 0.5 mM and 20 μM, respectively, at the pH and temperature optima (pH 9 and 30°C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.     

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from aKlebsiella pneumoniae mutant strain

Unlike the parent wild-type strain, theKlebsiella pneumoniae mutant strain MAO4 has a 4-HBA⁺ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when theAcinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoateK. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.     

Expression of a neu/c-erbB-2-like product in neuroendocrine cells of mammals

The neu/c-erbB-2 oncogene encodes a 185 kDa protein closely homologous to the epidermal growth factor receptor. The protein product (p185) is a glycoprotein with an external domain and an internal domain with tyrosine kinase activity. Amplification and/or overexpression of p185 is related to several human adenocarcinomas. Subsequent studies demonstrated its presence in certain neuroendocrine (NE) neoplasms, including phaeochromocytomas, insulinomas and medullary thyroid carcinomas. However, relatively little is known about its role in normal cell growth regulation and development. Therefore, our objective was to determine whether neu/c-erbB-2 was expressed in normal NE tissues of different mammals, specially in humans, as it was in their neoplasms. We have examined by immunohistochemistry different endocrine glands (thyroid, pancreas, suprarrenal and hypophysis) and the small intestine of human beings, rats and guinea pigs, using two polyclonal antibodies raised against the intracytoplasmic part of the protein, and specific antigen absorption controls. We have found that a neu/c-erbB-2-like product occurs in all normal NE tissues examined: C cells of the thyroid gland, chromaffin cells of the adrenal medulla, pancreatic islets, enteroendocrine cells of the small intestine and, finally, scattered cells of the adenohypophysis, according to a typical granular immunohistochemical pattern. Our results indicate that normal NE cells share a new common antigen in their cytoplasms, a neu/c-erbB-2-like product, with a similar immunostaining pattern to that presented by the neoplasms derived from them.     

Neospora caninum and Toxoplasma gondii antibodies in dogs from Durango City, Mexico

Toxoplasma gondii and Neospora caninum are structurally similar parasites, with many hosts in common. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs from Durango City, Mexico. Using a modified agglutination test, antibodies to T. gondii were found in 52 (51.5%) of the 101 dogs with titers of 1:25 in 27, 1:50 in 11, 1:100 in 5, 1:200 in 4, 1:400 in 2, 1:800 in 2, and 1:3,200 or higher in 1. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT) and the Neospora sp. agglutination test (NAT). Two of the 101 dogs had N. caninum antibodies; these dogs did not have T. gondii antibodies, supporting the specificity of the tests used. The N. caninum antibody titers of the 2 dogs were: 1:400 by IFAT and 1:200 by NAT in 1, and 1:25 by NAT and IFAT in the other. Results indicate that these 2 structurally similar protozoans are antigenically different.     

Cell envelope changes in Bifidobacterium animalis ssp. lactis as a response to bile

Bifidobacterium animalis ssp. lactis is a probiotic frequently used as adjunct culture in fermented dairy products. In order to ensure its proper function at the intestinal level, this bacterium has to be tolerant to physiological concentrations of bile. This study examined the influence of bile on the fatty acid composition and the membrane characteristics of B. animalis IPLA 4549 and its mutant with acquired resistance to bile, B. animalis 4549dOx. Bile adaptation triggers in B. animalis 4549dOx a decrease in membrane fluidity and in the protein : phospholipid ratio, as well as a shift in the fatty acid composition of the cell. Remarkably, the presence of bile in the growth medium induced similar changes in both B. animalis cells. Furthermore, transmission electron microscopy analysis showed that bile promotes a severe distortion of the cell surface. This study provides new insights of the action of bile on the cell envelope of bifidobacteria.     

Rapid conversion of spirostans into furostan skeletons at room temperature

We report a facile protocol to obtain 22-substituted furostans and pseudosapogenins in high yields from (25R)- and (25S)-sapogenins. This method involves the treatment of the sapogenin with acetic-trifluoroacetic mixed anhydride and BF(3)·OEt(2) at room temperature, followed by the addition of a nucleophile (H(2)O, MeOH or KSeCN). In the case of 22-hydroxyfurostans, they can be transformed to pseudosapogenins by treatment with p-toluensulfonic acid.     

Interspecific Introgression in Cetaceans: DNA Markers Reveal Post-F1 Status of a Pilot Whale

Visual species identification of cetacean strandings is difficult, especially when dead specimens are degraded and/or species are morphologically similar. The two recognised pilot whale species (Globicephala melas and Globicephala macrorhynchus) are sympatric in the North Atlantic Ocean. These species are very similar in external appearance and their morphometric characteristics partially overlap; thus visual identification is not always reliable. Genetic species identification ensures correct identification of specimens. Here we have employed one mitochondrial (D-Loop region) and eight nuclear loci (microsatellites) as genetic markers to identify six stranded pilot whales found in Galicia (Northwest Spain), one of them of ambiguous phenotype. DNA analyses yielded positive amplification of all loci and enabled species identification. Nuclear microsatellite DNA genotypes revealed mixed ancestry for one individual, identified as a post-F1 interspecific hybrid employing two different Bayesian methods. From the mitochondrial sequence the maternal species was Globicephala melas. This is the first hybrid documented between Globicephala melas and G. macrorhynchus, and the first post-F1 hybrid genetically identified between cetaceans, revealing interspecific genetic introgression in marine mammals. We propose to add nuclear loci to genetic databases for cetacean species identification in order to detect hybrid individuals.