Genetic identification of AChE as a positive modulator of addiction to the psychostimulant D-amphetamine in zebrafish

Addiction is a complex maladaptive behavior involving alterations in several neurotransmitter networks. In mammals, psychostimulants trigger elevated extracellular levels of dopamine, which can be modulated by central cholinergic transmission. Which elements of the cholinergic system might be targeted for drug addiction therapies remains unknown. The rewarding properties of drugs of abuse are central for the development of addictive behavior and are most commonly measured by means of the conditioned place preference (CPP) paradigm. We demonstrate here that adult zebrafish show robust CPP induced by the psychostimulant D-amphetamine. We further show that this behavior is dramatically reduced upon genetic impairment of acetylcholinesterase (AChE) function in ache/+ mutants, without involvement of concomitant defects in exploratory activity, learning, and visual performance. Our observations demonstrate that the cholinergic system modulates drug-induced reward in zebrafish, and identify genetically AChE as a promising target for systemic therapies against addiction to psychostimulants. More generally, they validate the zebrafish model to study the effect of developmental mutations on the molecular neurobiology of addiction in vertebrates.     

Phosphatase Activity Toward Abnormally Phosphorylated τ: Decrease in Alzheimer Disease Brain

Abstract: Microtubule-associated protein τ is abnormally hyperphosphorylated and aggregated in affected neurons of Alzheimer disease brain. This hyperphosphorylated τ can be dephosphorylated at some of the abnormal phosphorylated sites by purified protein phosphatase-1, 2A, and 2B in vitro. In the present study, we have developed an assay to measure protein phosphatase activity toward τ-1 sites (Ser¹⁹⁹/Ser²⁰²) using the hyperphosphorylated τ isolated from Alzheimer disease brain as substrate. Using this assay, we have identified that in normal brain, protein phosphatase-2A and 2B and, to a lesser extent, 1 are involved in the dephosphorylation of τ. The K _m values of dephosphorylation of the hyperphosphorylated τ by protein phosphatase-2A and 2B are similar. The τ phosphatase activity is decreased by ∼30% in brain of Alzheimer disease patients compared with those of age-matched controls. These findings suggest that a defect of protein phosphatase could be the cause of the abnormal hyperphosphorylation of τ in Alzheimer disease.     

Effect of alemtuzumab-based T-cell depletion on graft compositional change in vitro and immune reconstitution early after allogeneic stem cell transplantation

Background aimsTo reduce the risk of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT), T-cell depletion (TCD) of grafts can be performed by the addition of alemtuzumab (ALT) “to the bag” (in vitro) before transplantation. In this prospective study, the authors analyzed the effect of in vitro incubation with 20 mg ALT on the composition of grafts prior to graft infusion. Furthermore, the authors assessed whether graft composition at the moment of infusion was predictive for T-cell reconstitution and development of GVHD early after TCD alloSCT.MethodsSixty granulocyte colony-stimulating factor-mobilized stem cell grafts were obtained from ≥9/10 HLA-matched related and unrelated donors. The composition of the grafts was analyzed by flow cytometry before and after in vitro incubation with ALT. T-cell reconstitution and incidence of severe GVHD were monitored until 12 weeks after transplantation.ResultsIn vitro incubation of grafts with 20 mg ALT resulted in an initial median depletion efficiency of T-cell receptor (TCR) α/β T cells of 96.7% (range, 63.5–99.8%), followed by subsequent depletion in vivo. Graft volumes and absolute leukocyte counts of grafts before the addition of ALT were not predictive for the efficiency of TCR α/β T-cell depletion. CD4^pos T cells were depleted more efficiently than CD8^pos T cells, and naive and regulatory T cells were depleted more efficiently than memory and effector T cells. This differential depletion of T-cell subsets was in line with their reported differential CD52 expression. In vitro depletion efficiencies and absolute numbers of (naive) TCR α/β T cells in the grafts after ALT incubation were not predictive for T-cell reconstitution or development of GVHD post- alloSCT.ConclusionsThe addition of ALT to the bag is an easy, fast and generally applicable strategy to prevent GVHD in patients receiving alloSCT after myeloablative or non-myeloablative conditioning because of the efficient differential depletion of donor-derived lymphocytes and T cells.     

The use of genetic markers to measure genomic response to selection in livestock

A method to measure genomic response to natural and artificial selection by means of genetic markers in livestock is proposed. Genomic response through several levels of selection was measured using sequential testing for distorted segregation of alleles among selected and nonselected sons, single-sperm typing, and a test with records for growth performance. Statistical power at a significance level of 0.05 was >0.5 for a marker linked to a QTL with recombination fractions 0, 0.10, and 0.20 for detecting genomic responses for gene effects of 0.6, 0.7, and 1.0 phenotypic standard deviations, respectively. Genomic response to artificial selection in six commercial bull sire families comprising 285 half-sib sons selected for growth performance was measured using 282 genetic markers evenly distributed over the cattle genome. A genome-wide test using selected sons was significant (P < 0.001), indicating that selection induces changes in the genetic makeup of commercial cattle populations. Markers located in chromosomes 6, 10, and 16 identified regions in those chromosomes that are changing due to artificial selection as revealed by the association of records of performance with alleles at specific markers. Either natural selection or genetic drift may cause the observed genomic response for markers in chromosomes 1, 7, and 17.     

Energy-dependent accumulation of iron by isolated rat liver mitochondria V. Effect on factors controlling respiration and oxidative phosphorylation

1. Depending on the metabolic state, the addition of iron(III)-sucrose induces an inhibition or a stimulation of the respiration rate when added to isolated rat liver mitochondria.2. Under conditions identical to those used in the accumulation studies (Romslo, I. and Flatmark, T. (1973) Biochim. Biophys. Acta 305, 29?40), the ferric complex induces a decrease in the oxygen uptake concomitant to an oxidation of cytochromes c (+c₁) and a (+a₃). These results suggest that ferric iron is reduced to ferrous iron by the respiratory chain prior to or simultaneously with its energy-dependent accumulation.3. On the other hand, the addition of iron(III)-sucrose induces a stimulation of respiration in State 4 and State 3 provided Mg²⁺ is present in the suspending medium. In contrast to Ca²⁺, iron stimulates State 4 respiration in a cyclic process only within narrow concentration limits; at concentrations of iron above 100 μM the respiration remains in the activated state until anaerobiosis. The stimulation of State 4 respiration is more pronounced with succinate than with NAD-linked substrates, a difference which partly may be attributed to a stimulation of the succinate dehydrogenase complex.4. The stimulation of respiration by iron is approx. 3 times higher in State 3 than in State 4 and this difference can be attributed to a stimulation of the adenine nucleotide exchange reaction in State 3 with a concomitant increase in the rate of oxidative phosphorylation, although the $\text{P}\text{O}$ ratio is slightly diminished.     

Visualization of the target-membrane-inserted fusion protein of Semliki Forest virus by combined electron microscopy and crystallography

Semliki Forest virus enters cells by receptor-mediated endocytosis. The acidic environment of the endosome triggers a membrane fusion reaction that is mediated by the E1 glycoprotein. During fusion, E1 rearranges from an E1/E2 heterodimer to a highly stable, membrane-inserted E1 homotrimer (E1HT). In this study, we analyzed E1HT by a combination of electron cryomicroscopy, electron crystallography of negatively stained 2D crystals, and fitting of the available X-ray structure of the monomeric E1 ectodomain into the resulting 3D reconstruction. The visualized E1HT reveals that the ectodomain has reoriented vertically and inserted the distal tip of domain II into the lipid bilayer. Our data allow the visualization of a viral fusion protein inserted in its target membrane and demonstrate that insertion is a cooperative process, resulting in rings composed of five to six homotrimers.     

The prevalence of smoking among Croatian hospitalized coronary heart disease patients

The aim of this paper was to investigate the prevalence of smoking using selected anthropometric variables in a sample of hospitalized coronary heart disease (CHD) patients in Croatia (N = 1,298). A total of 444 subjects (34.6%) were non-smokers, 548 (42.6%) were smokers and 293 (22.8%) were ex-smokers. Men, on average, smoked more cigarettes per day than women (22.62 vs. 19.84 cigarettes, p < 0.001) and they also had bigger index "pack-years" than women (36.96 vs. 33.91, p = 0.024). Men were more often smokers and ex-smokers than women (47.4% vs. 30.8% for smokers and 25.0% vs. 22.8% for ex-smokers, p < 0.001). In this study a high prevalence of smoking was found among CHD patients in Croatia. Unless it is decreased, it can be expected that CHD patients in Croatia will continue to experience adverse effects more often than other CHD patients in the rest of Europe.