Overexpression of the CHRNA5/A3/B4 genomic cluster in mice increases the sensitivity to nicotine and modifies its reinforcing effects

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated pentameric ion channels that account for the effects of nicotine. Recent genetic studies have highlighted the importance of variants of the CHRNA5/A3/B4 genomic cluster in human nicotine dependence. Among these genetic variants those found in non-coding segments of the cluster may contribute to the pathophysiology of tobacco use through alterations in the expression of these genes. To discern the in vivo effects of the cluster, we generated a transgenic mouse overexpressing the human CHRNA5/A3/B4 cluster using a bacterial artificial chromosome. Transgenic mice showed increased functional α3β4-nAChRs in brain regions where these subunits are highly expressed under normal physiological conditions. Moreover, they exhibited increased sensitivity to the pharmacological effects of nicotine along with higher activation of the medial habenula and reduced activation of dopaminergic neurons in the ventral tegmental area after acute nicotine administration. Importantly, transgenic mice showed increased acquisition of nicotine self-administration (0.015 mg/kg per infusion) and a differential response in the progressive ratio test. Our study provides the first in vivo evidence of the involvement of the CHRNA5/A3/B4 genomic cluster in nicotine addiction through modifying the activity of brain regions responsible for the balance between the rewarding and the aversive properties of this drug.     

Experimental litter size reduction reveals costs of gestation and delayed effects on offspring in a viviparous lizard

Experimental studies have often been employed to study costs of reproduction, but rarely to study costs of gestation. Disentangling the relative importance of each stage of the reproductive cycle should help to assess the costs and benefits of different reproductive strategies. To that end, we experimentally reduced litter size during gestation in a viviparous lizard. We measured physiological and behavioural parameters during gestation and shortly after parturition, as well as survival and growth of females and their offspring. This study showed four major results. First, the experimental litter size reduction did not significantly affect the cellular immune response, the metabolism and the survival of adult females. Second, females with reduced litter size decreased their basking time. Third, these females also had an increased postpartum body condition. As postpartum body condition is positively related to future reproduction, this result indicates a gestation cost. Fourth, even though offspring from experimentally reduced litters had similar weight and size at birth as other offspring, their growth rate after birth was significantly increased. This shows the existence of a maternal effect during gestation with delayed consequences. This experimental study demonstrates that there are some costs to gestation, but it also suggests that some classical trade-offs associated with reproduction may not be explained by gestation costs.     

Abundance and chromosomal distribution of six Drosophila buzzatii transposons: BuT1, BuT2, BuT3, BuT4, BuT5, and BuT6

The abundance and chromosomal distribution of six class-II transposable elements (TEs) of Drosophila buzzatii have been analyzed by Southern blotting and in situ hybridization. These six transposons had been previously found at the breakpoints of inversions 2j and 2q ⁷ of D. buzzatii. These two polymorphic inversions were generated by an ectopic recombination event between two copies of Galileo, a Foldback element. The four breakpoints became hotspots for TE insertions after the generation of the inversion and the transposons analyzed in this work are considered to be secondary invaders of these regions. Insertions of the six transposons are present in the euchromatin but show an increased density in the pericentromeric euchromatin–heterochromatin transition region and the dot chromosome. They are also more abundant in the inverted segments of chromosome 2 rearrangements. We further observed that the accumulation of TE insertions varies between elements and is correlated between dot, proximal regions, and inverted segments. These observations fully agree with previous data in Drosophila melanogaster and support recombination rate as the chief force explaining the chromosomal distribution of TEs.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.Sequence data from this article have been deposited in the EMBL/GenBank Data Libraries under accession number DQ402469.     

Antioxidant activity in Spalax ehrenbergi: a possible adaptation to underground stress

The blind subterranean mole rat Spalax ehrenbergi superspecies has evolved adaptive strategies to cope with underground stress. Hypoxia is known to stimulate reactive oxygen species generation; however, mechanisms by which Spalax counteracts oxidative damage have not been investigated before. We studied in Spalax the oxidative status of the Harderian gland (HG), an organ which is particularly vulnerable to oxidative stress in many rodents. With regard to the sexual dimorphism found in this gland, differences between males and females were determined and compared to the surface-dwelling Syrian hamster. Our results show, for the first time, that Spalax exhibits remarkably low biomolecular damage, which implies the existence of physiological strategies to avoid oxidative damage under fluctuating O₂ and CO₂ levels existing in the mole rat’s subterranean niche. Correspondingly, main antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione reductase (GR), exhibited high activities in both genders; in particular, remarkably high levels were measured in SOD. SOD and GR activities showed statistically significant differences between sexes. Melatonin, an important circadian agent is also a very important antioxidant molecule and is synthesized in the Harderian glands (HGs) of Spalax. Therefore, the possible interaction between antioxidant enzymes and melatonin is suggested.Joint senior authorship: Aaron Avivi and Ana Coto-Montes     

Changes in hydrophilic antioxidant activity in Avena sativa and Triticum aestivum leaves of different age during de-etiolation and high-light treatment

The steady-state of reactive oxygen species (ROS) in plant cells is controlled by ROS-producing and scavenging agents. A large cellular pool of antioxidant metabolites is involved in their control. Variations in this antioxidant pool may be monitored by measuring changes in hydrophilic antioxidant activity (free radical-quenching activity of water-soluble components) and ascorbic acid levels. The de-etiolation process and induction of light stress in Avena sativa and Triticum aestivum leaves were used as physiological models to study the antioxidant status at different ages. The data showed that five-day-old green plants and de-etiolated plants of the same age have similar hydrophilic antioxidant activity (8 mol ASC equivalents g FW^–1), which increases during the de-etiolation process. In oat and wheat, young leaves (five days old) had higher antioxidant status (hydrophilic antioxidant activity and ascorbic acid level) than old leaves (10 and 20 days old). High-light treatment caused a decrease in antioxidant status, especially in young leaves. Hydrophilic antioxidant activity and ascorbic acid levels recovered totally or partially after 30 or 60 min in the dark. This capacity also depends on age and species. The ascorbic acid/hydrophilic antioxidant activity ratio is presented as an indicator of antioxidant variations in response to stress, but taking into account the absolute levels of antioxidants.     

Membrane remodelling during vaccinia virus morphogenesis

Background information. VACV (vaccinia virus) is one of the most complex viruses, with a size exceeding 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and important rearrangements of its structural components. Results. We have used electron tomography of sections of VACV‐infected cells to follow, in three dimensions, the remodelling of the membrane components of the virus during envelope maturation. The tomograms obtained suggest that a number of independent ‘crescents’ interact with each other to enclose the volume of an incomplete ellipsoid in the viral factory area, attaining the overall shape and size characteristic of the first immature form of the virus [IV (immature virus)]. The incorporation of the DNA into these forms leads to particles with a nucleoid [IVN (IV with nucleoid)] that results in local disorganization of the envelope in regions near the condensed DNA. These particles suffer the progressive disappearance of the membrane outer spikes with a change in the shape of the membrane, becoming locally curled. The transformation of the IVN into the mature virus involves an extreme rearrangement of the particle envelope, which becomes fragmented and undulated. During this process, we also observed connections between the outer membranes with internal ones, suggesting that the latter originate from internalization of the IV envelope. Conclusions. The main features observed for VACV membrane maturation during morphogenesis resemble the breakdown and reassembly of cellular endomembranes.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.     

Hexadecylphosphocholine alters nonvesicular cholesterol traffic from the plasma membrane to the endoplasmic reticulum and inhibits the synthesis of sphingomyelin in HepG2 cells

The synthetic lipid analogue, hexadecylphosphocholine is an antitumoral and antileishmanial agent that acts on cell membranes and can induce apoptosis. We have previously investigated the effect of hexadecylphosphocholine on the biosynthesis and intracellular transport of cholesterol in the human hepatoma HepG2 cell line. Here we show that the traffic of endocytosed-cholesterol from LDL to the plasma membrane and the transport of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane were unaffected by alkylphosphocholine exposure. On the contrary, cholesterol traffic from the plasma membrane to the endoplasmic reticulum was drastically interrupted after 1 h of cell exposition to HePC and, consequently, the intracellular esterification of cholesterol was substantially decreased. Our results also demonstrate that this alkylphosphocholine exclusively affected the nonvesicular, energy-independent cholesterol traffic, without altering the vesicular transport. In addition, hydrolysis of plasma membrane sphingomyelin by exogenously added sphingomyelinase resulted in enhanced plasma-membrane cholesterol esterification, but sphingomyelinase treatment did not prevent the inhibition in cholesteryl ester formation caused by hexadecylphosphocholine. We also found that sphingomyelin synthesis was significantly inhibited in HepG2 cells after exposure to hexadecylphosphocholine. Since sphingomyelin and cholesterol are major lipid constituents of membrane raft microdomains, these results suggest that hexadecylphosphocholine could disturb membrane raft integrity and thence its functionality.