Synthesis and structure-activity relationship of 2-(aminoalkyl)-2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine derivatives: a novel series of 5-HT(2A/2C) receptor antagonists. Part 2.

Following the programme started at Janssen Research Foundation searching for 5-HT(2A/2C) antagonists, we now report on the synthesis of a series of substituted 2-(Dimethylaminomethyl)-2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine derivatives. The 5-HT(2A), 5-HT(2C) and H(1) receptor affinities as well as the mCPP antagonistic activity of the compounds synthesised is described.     

P transposition in Drosophila provides a new tool for analyzing postreplication repair and double-strand break repair.

A genetic screen has been developed in Drosophila for identifying host-repair genes responsible for processing DNA lesions formed during mobilization of P transposable elements. Application of that approach to repair deficient mutants has revealed that the mei-41 and mus302 genes are necessary for recovery of P-bearing chromosomes undergoing transposition. Both of these genes are required for normal postreplication repair. Mutants deficient in excision repair, on the other hand, have no detected effect on the repair of transposition-induced lesions. These observations suggest that P element-induced lesions are repaired by a postreplication pathway of DNA repair. The data further support recent studies implicating double-strand DNA breaks as intermediates in P transposition, because the mei-41 gene has been genetically and cytologically associated with the repair of interrupted chromosomes. Analysis of this system has also revealed a striking stimulation of site-specific gene conversion and recombination by P transposition. This result strongly suggests that postreplication repair in this model eukaryote operates through a conversion/recombination mechanism. Our results also support a recently developed model for a conversion-like mechanism of P transposition (Engels et al., 1990). Involvement of the mei-41 and mus302 genes in the repair of P element-induced double-strand breaks and postreplication repair points to a commonality in the mechanisms of these processes.     

A survey of porcine kidneys and chicken liver for ochratoxin A in Spain

Contamination studies by ochratoxin A on pork kidney and chicken liver has been carried out in Catalonia (Spain). 73% of the pork kidney samples analyzed did not contain an amount of ochratoxin A over our detection limit (0.5 ng/g) whereas only 7% had contamination higher than 1 ng/g. None of the chicken samples analyzed were contaminated by this toxin above the detection limit. All contamination levels found are below the maximum levels accepted by several countries for this kind of material. A confirmative test is necessary before discarding false positive samples.     

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.     

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.     

Engineering E. coli for improved microaerobic pDNA production

Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA⁻, which, together with W3110recA⁻vgb⁺, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production.

     

Lifelong choline supplementation ameliorates Alzheimer’s disease pathology and associated cognitive deficits by attenuating microglia activation

Currently, there are no effective therapies to ameliorate the pathological progression of Alzheimer's disease (AD). Evidence suggests that environmental factors may contribute to AD. Notably, dietary nutrients are suggested to play a key role in mediating mechanisms associated with brain function. Choline is a B‐like vitamin nutrient found in common foods that is important in various cell functions. It serves as a methyl donor and as a precursor for production of cell membranes. Choline is also the precursor for acetylcholine, a neurotransmitter which activates the alpha7 nicotinic acetylcholine receptor (α7nAchR), and also acts as an agonist for the Sigma‐1 R (σ1R). These receptors regulate CNS immune response, and their dysregulation contributes to AD pathogenesis. Here, we tested whether dietary choline supplementation throughout life reduces AD‐like pathology and rescues memory deficits in the APP/PS1 mouse model of AD. We exposed female APP/PS1 and NonTg mice to either a control choline (1.1 g/kg choline chloride) or a choline‐supplemented diet (5.0 g/kg choline chloride) from 2.5 to 10 months of age. Mice were tested in the Morris water maze to assess spatial memory followed by neuropathological evaluation. Lifelong choline supplementation significantly reduced amyloid‐β plaque load and improved spatial memory in APP/PS1 mice. Mechanistically, these changes were linked to a decrease of the amyloidogenic processing of APP, reductions in disease‐associated microglial activation, and a downregulation of the α7nAch and σ1 receptors. Our results demonstrate that lifelong choline supplementation produces profound benefits and suggest that simply modifying diet throughout life may reduce AD pathology.     

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.     

Structural characterization of beta-cardiac myosin subfragment 1 in solution

beta-cardiac myosin subfragment 1 (betaS1) tertiary structure and dynamics were characterized with proteolytic digestion, nucleotide analogue trapping kinetics, and intrinsic fluorescence changes accompanying nucleotide binding. Proteolysis of betaS1 produces the 25, 50, and 20 kDa fragments and a new cut within the 50-kDa fragment at Arg369. F-actin inhibits cleavage of the 50-kDa fragment and fails to inhibit cleavage at the 50/20 kDa junction, suggesting betaS1 presents an actoS1 conformation fundamentally different from skeletal S1. Time-dependent changes in Mg(2+)-ATPase accompanying proteolysis identifies cleavage points that lie within the energy transduction pathway. The nucleotide analogue trapping kinetics reveal the presence of a reversible weakly actin attached state. Comparison of nucleotide analogue induced betaS1 structures with the transient structures occurring during ATPase indicates analogue induced and transient structures are in a one-to-one correspondence. Tryptophan fluorescence enhancement accompanies the binding or trapping of nucleotide or nucleotide analogues. Isolation of Trp508 fluorescence shows it is an ATP-sensitive tryptophan and that its vicinity changes conformation sequentially with the transient intermediates accompanying ATPase. These studies elucidate energy transduction and suggest how mutations of betaS1 implicated in disease might undermine function, stability, or efficiency.     

The control of seizure-like activity in the rat hippocampal slice

The sudden and transient hypersynchrony of neuronal firing that characterizes epileptic seizures can be considered as the transitory stabilization of metastable states present within the dynamical repertoire of a neuronal network. Using an in vitro model of recurrent spontaneous seizures in the rat horizontal hippocampal slice preparation, we present an approach to characterize the dynamics of the transition to seizure, and to use this information to control the activity and avoid the occurrence of seizure-like events. The transition from the interictal activity (between seizures) to the seizure-like event is aborted by brief (20-50 s) low-frequency (0.5 Hz) periodic forcing perturbations, applied via an extracellular stimulating electrode to the mossy fibers, the axons of the dentate neurons that synapse onto the CA3 pyramidal cells. This perturbation results in the stabilization of an interictal-like low-frequency firing pattern in the hippocampal slice. The results derived from this work shed light on the dynamics of the transition to seizure and will further the development of algorithms that can be used in automated devices to stop seizure occurrence.