Identification of Sare0718 As an Alanine-Activating Adenylation Domain in Marine Actinomycete Salinispora arenicola CNS-205

BACKGROUND: Amino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research. Salinispora arenicola CNS-205 is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate cryptic NRP-related metabolites in S. arenicola CNS-205, we cloned and identified the putative gene sare0718 annotated "amino acid adenylation domain". Firstly, the general features and possible functions of sare0718 were predicted by bioinformatics analysis, which suggested that Sare0718 is a soluble protein with an AMP-binding domain contained in the sequence and its cognate substrate is L-Val. Then, a GST-tagged fusion protein was expressed and purified to further explore the exact adenylation activity of Sare0718 in vitro. By a newly mentioned nonradioactive malachite green colorimetric assay, we found that L-Ala but not L-Val is the actual activated amino acid substrate and the basic kinetic parameters of Sare0718 for it are K(m) = 0.1164±0.0159 (mM), V(max) = 3.1484±0.1278 (µM/min), k(cat) = 12.5936±0.5112 (min(-1)). CONCLUSIONS/SIGNIFICANCE: By revealing the biochemical role of sare0718 gene, we identified an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205, which would provide useful information for next isolation and function elucidation of the whole cryptic nonribosomal peptide synthetase (NRPS)-related gene cluster covering Sare0718. And meanwhile, this work also enriched the biochemical data of A domain substrate specificity in newly discovered marine actinomycete NRPS system, which bioinformatics prediction will largely depend on

A Barcode Screen for Epigenetic Regulators Reveals a Role for the NuB4/HAT-B Histone Acetyltransferase Complex in Histone Turnover

Dynamic modification of histone proteins plays a key role in regulating gene expression. However, histones themselves can also be dynamic, which potentially affects the stability of histone modifications. To determine the molecular mechanisms of histone turnover, we developed a parallel screening method for epigenetic regulators by analyzing chromatin states on DNA barcodes. Histone turnover was quantified by employing a genetic pulse-chase technique called RITE, which was combined with chromatin immunoprecipitation and high-throughput sequencing. In this screen, the NuB4/HAT-B complex, containing the conserved type B histone acetyltransferase Hat1, was found to promote histone turnover. Unexpectedly, the three members of this complex could be functionally separated from each other as well as from the known interacting factor and histone chaperone Asf1. Thus, systematic and direct interrogation of chromatin structure on DNA barcodes can lead to the discovery of genes and pathways involved in chromatin modification and dynamics

Memantine shows promise in reducing gambling severity and cognitive inflexibility in pathological gambling: a pilot study

RATIONALE: Although pathological gambling (PG) is relatively common, pharmacotherapy research for PG is limited. Memantine, an N-methyl d-aspartate receptor antagonist, appears to reduce glutamate excitability and improve impulsive decision making, suggesting it may help individuals with PG. OBJECTIVE: This study sought to examine the safety and efficacy of Memantine in PG. METHODS: Twenty-nine subjects (18 females) with DSM-IV PG were enrolled in a 10-week open-label treatment study of memantine (dose ranging from 10 to 30 mg/day). Subjects were enrolled from January 2009 until April 2010. Change from baseline to study endpoint on the Yale Brown Obsessive Compulsive Scale Modified for Pathological Gambling (PG-YBOCS) was the primary outcome measure. Subjects underwent pre- and post-treatment cognitive assessments using the stop-signal task (assessing response impulsivity) and the intra-dimensional/extra-dimensional (ID/ED) set shift task (assessing cognitive flexibility). RESULTS: Twenty-eight of the 29 subjects (96.6%) completed the 10-week study. PG-YBOCS scores decreased from a mean of 21.8±4.3 at baseline to 8.9±7.1 at study endpoint (p<0.001). Hours spent gambling per week and money spent gambling both decreased significantly (p<0.001). Subjects also demonstrated a significant improvement in ID/ED total errors (p=0.037) at study endpoint. The mean effective dose of memantine was 23.4±8.1 mg/day. The medication was well-tolerated. Memantine treatment was associated with diminished gambling and improved cognitive flexibility. CONCLUSIONS: These findings suggest that pharmacological manipulation of the glutamate system may target both gambling and cognitive deficits in PG. Placebo-controlled, double-blind studies are warranted in order to confirm these preliminary findings in a controlled design