Characterization of the recombinant diaminobutyric acid acetyltransferase from Methylophaga thalassica and Methylophaga alcalica

Diaminobutyric acid acetyltransferase (EctA) catalyzes the acetylation of diaminobutyric acid to gamma-N-acetyl-alpha,gamma-diaminobutyrate with acetyl coenzyme A. This is the second reaction in the ectoine biosynthetic pathway. The recombinant EctA proteins were purified from two moderately halophilic methylotrophic bacteria: Methylophaga thalassica ATCC 33146T and Methylophaga alcalica ATCC 35842T. EctA found in both methylotrophs is a homodimer with a subunit molecular mass of c. 20 kDa and had similar properties with respect to the optimum temperature for activity (30 degrees C), Km for diaminobutyrate (370 or 375 microM) and the absence of requirements for divalent metal ions. The enzyme from M. thalassica exhibited a lower pH optimum and was inhibited both by sodium carbonates and by high ionic strength but to a lesser extent by copper ions.     

Pten deletion causes mTorc1-dependent ectopic neuroblast differentiation without causing uniform migration defects

Neuronal precursors, generated throughout life in the subventricular zone, migrate through the rostral migratory stream to the olfactory bulb where they differentiate into interneurons. We found that the PI3K-Akt-mTorc1 pathway is selectively inactivated in migrating neuroblasts in the subventricular zone and rostral migratory stream, and activated when these cells reach the olfactory bulb. Postnatal deletion of Pten caused aberrant activation of the PI3K-Akt-mTorc1 pathway and an enlarged subventricular zone and rostral migratory stream. This expansion was caused by premature termination of migration and differentiation of neuroblasts and was rescued by inhibition of mTorc1. This phenotype is reminiscent of lamination defects caused by Pten deletion in developing brain that were previously described as defective migration. However, live imaging in acute slices showed that Pten deletion did not cause a uniform defect in the mechanics of directional neuroblast migration. Instead, a subpopulation of Pten-null neuroblasts showed minimal movement and altered morphology associated with differentiation, whereas the remainder showed unimpeded directional migration towards the olfactory bulb. Therefore, migration defects of Pten-null neurons might be secondary to ectopic differentiation.     

Morphological aspects of the hypothalamic-hypophyseal system

Summary The vascularity of the hypothalamus in rats, cats, and dogs was studied with various injection methods. Particular attention was paid to the capillary architecture of the hypothalamus. The density of the capillary bed in the nuclei of the anterior and medial hypothalamus was studied in male albino rats after filling the cerebral vessels with 5 per cent India ink—gelatin mass. The data obtained were compared with the magnitude-S (projection areas of the capillary meshes of the three-dimensional meshwork) distributions and with the calibres of the capillaries. Distinct inverse correlations were demonstrated between these indices. The objective tests used provided evidence that the peculiar features of the capillary architecture in the various nuclei of the anterior and medial hypothalamus are closely connected with the degree of their vascularity. The rich vascularity of the neurosecretory nuclei is discussed in connection with the peculiarities of their metabolism.The author wishes to express his sincere gratitude to Prof. S. M. Blinkov for valuable advice, to Dr. V. I. Prilutsky for the help received from him in biometric treatment and mathematical analysis of the data obtained, and to Natalia Samushkina and Galina Yankova for their helpful technical assistance.     

Strengths and limitations of genetic models of ADHD

The cause and pathophysiology of attention-deficit hyperactivity disorder (ADHD) are unknown, but compelling evidence suggests an involvement of genetic factors. While dopamine is believed to play a major role in ADHD, the role for norepinephrine and serotonin systems has also been indicated. Mutant mice are valuable tools to dissect the contribution of specific neurotransmitter systems to brain dysfunction and particularly useful to decode complex multi-transmitter interaction that is critical to the pathophysiology of ADHD. Genetically altered mice provided also an opportunity to test experimentally the role of novel candidate genes for this disorder identified in genetic clinical studies. While it is clear that no rodent model would be able to recapitulate fully the complex nature of ADHD, certain endophenotypes could be reasonably well mimicked in these models. Multiple studies have reported associations between polymorphisms in dopamine transporter (DAT) gene and ADHD. Although the functional consequences of these associations are still unclear, it is believed that alterations in DAT-mediated processes might contribute to the pathogenesis of ADHD. Mice lacking the dopamine transporter have elevated dopaminergic tone and represent a genetic animal model in which certain endophenotypes of ADHD can be recapitulated. These mutants as well as other mouse models of DAT dysfunction provided an opportunity to investigate the neuronal circuitry and molecular mechanisms involved in the inhibitory action of psychostimulants on hyperactivity. Several additional knockout and transgenic mouse models have been proposed to model ADHD. Strengths and limitations of currently available genetic mouse models of ADHD are discussed.     

Reduced expression of the vesicular acetylcholine transporter causes learning deficits in mice

Storage of acetylcholine in synaptic vesicles plays a key role in maintaining cholinergic function. Here we used mice with a targeted mutation in the vesicular acetylcholine transporter (VAChT) gene that reduces transporter expression by 40% to investigate cognitive processing under conditions of VAChT deficiency. Motor skill learning in the rotarod revealed that VAChT mutant mice were slower to learn this task, but once they reached maximum performance they were indistinguishable from wild-type mice. Interestingly, motor skill performance maintenance after 10 days was unaffected in these mutant mice. We also tested whether reduced VAChT levels affected learning in an object recognition memory task. We found that VAChT mutant mice presented a deficit in memory encoding necessary for the temporal order version of the object recognition memory, but showed no alteration in spatial working memory, or spatial memory in general when tested in the Morris water maze test. The memory deficit in object recognition memory observed in VAChT mutant mice could be reversed by cholinesterase inhibitors, suggesting that learning deficits caused by reduced VAChT expression can be ameliorated by restoring ACh levels in the synapse. These data indicate an important role for cholinergic tone in motor learning and object recognition memory.     

A regulatory domain in the N terminus of tryptophan hydroxylase 2 controls enzyme expression

Serotonin is involved in a variety of physiological processes in the central nervous system and the periphery. As the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase plays an important role in modulating these processes. Of the two variants of tryptophan hydroxylase, tryptophan hydroxylase 2 (TPH2) is expressed predominantly in the central nervous system, whereas tryptophan hydroxylase 1 (TPH1) is expressed mostly in peripheral tissues. Although the two enzymes share considerable sequence homology, the regulatory domain of TPH2 contains an additional 41 amino acids at the N terminus that TPH1 lacks. Here we show that the extended TPH2 N-terminal domain contains a unique sequence involved in the regulation of enzyme expression. When expressed in cultured mammalian cells, TPH2 is synthesized less efficiently and is also less stable than TPH1. Removal of the unique portion of the N terminus of TPH2 results in expression of the enzyme at a level similar to that of TPH1, whereas protein chimeras containing this fragment are expressed at lower levels than their wild-type counterparts. We identify a region centered on amino acids 10-20 that mediates the bulk of this effect. We also demonstrate that phosphorylation of serine 19, a protein kinase A consensus site located in this N-terminal domain, results in increased TPH2 stability and consequent increases in enzyme output in cell culture systems. Because this domain is unique to TPH2, these data provide evidence for selective regulation of brain serotonin synthesis.     

Dopamine transporter-dependent and -independent actions of trace amine beta-phenylethylamine

Beta-phenylethylamine (beta-PEA) is an endogenous amine that is found in trace amounts in the brain. It is believed that the locomotor-stimulating action of beta-PEA, much like amphetamine, depends on its ability to increase extracellular dopamine (DA) concentrations owing to reversal of the direction of dopamine transporter (DAT)-mediated DA transport. beta-PEA can also bind directly to the recently identified G protein-coupled receptors, but the physiological significance of this interaction is unclear. To assess the mechanism by which beta-PEA mediates its effects, we compared the neurochemical and behavioral effects of this amine in wild type (WT), heterozygous and 'null' DAT mutant mice. In microdialysis studies, beta-PEA, administered either systemically or locally via intrastriatal infusion, produced a pronounced outflow of striatal DA in WT mice whereas no increase was detected in mice lacking the DAT (DAT-KO mice). Similarly, in fast-scan voltammetry studies beta-PEA did not alter DA release and clearance rate in striatal slices from DAT-KO mice. In behavioral studies beta-PEA produced a robust but transient increase in locomotor activity in WT and heterozygous mice. In DAT-KO mice, whose locomotor activity and stereotypy are increased in a novel environment, beta-PEA (10-100 mg/kg) exerted a potent inhibitory action. At high doses, beta-PEA induced stereotypies in WT and heterozygous mice; some manifestations of stereotypy were also observed in the DAT-KO mice. These data demonstrate that the DAT is required for the striatal DA-releasing and hyperlocomotor actions of beta-PEA. The inhibitory action on hyperactivity and certain stereotypies induced by beta-PEA in DAT-KO mice indicate that targets other than the DAT are responsible for these effects.     

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M(2) muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene (GRK5(-/-)) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5(-/-) and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from beta(2)-adrenergic receptor activation were diminished in GRK5(-/-) mice. These data suggest that M(2) muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5(-/-) mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.