Selective inhibition of human acetylcholinesterase by galanthamine in vitro and in vivo

We investigated the inhibition of human cholinesterases by galanthamine, an alkaloid of the common snowdrop (galanthus nivalis). In vitro, the compound showed potent enzyme inhibition and 50-fold selectivity for acetylcholinesterase (EC 3.1.1.7) as opposed to butyrylcholinesterase (EC 3.1.1.8). There was no difference between enzyme inhibition by galanthamine in whole blood and separated fractions of plasma and erythrocytes. We conclude that galanthamine does not accumulate in large amounts in red blood cells. In vivo, administration of galanthamine in a healthy volunteer and in a patient who underwent long-term treatment confirmed the selectivity of galanthamine for acetylcholinesterase.     

Induction of therapeutic abortion using either extra-amniotic prostaglandin F2α or hypertonic saline followed by oxytocin : A clinical comparison of the two methods

PGF_2α or hypertonic (20 per cent) saline followed by oxytocin was used to terminate pregnancy in 160 women between the 10th and 20th weeks of gestation.In the patients treated with PGF_2α minor side-effects were reported in about 50 per cent of the cases, however, the method was superior to hypertonic saline with regard to the number of complications and the length of stay in hospital.     

Genome-inferred spatio-temporal resolution of an uncultivated Roizmanbacterium reveals its ecological preferences in groundwater

Subsurface ecosystems like groundwater harbour diverse microbial communities, including small-sized, putatively symbiotic organisms of the Candidate Phyla Radiation, yet little is known about their ecological preferences and potential microbial partners. Here, we investigated a member of the superphylum Microgenomates (Cand. Roizmanbacterium ADI133) from oligotrophic groundwater using mini-metagenomics and monitored its spatio-temporal distribution using 16S rRNA gene analyses. A Roizmanbacteria-specific quantitative PCR assay allowed us to track its abundance over the course of 1 year within eight groundwater wells along a 5.4 km hillslope transect, where Roizmanbacteria reached maximum relative abundances of 2.3%. In-depth genomic analyses suggested that Cand. Roizmanbacterium ADI133 is a lactic acid fermenter, potentially able to utilize a range of complex carbon substrates, including cellulose. We hypothesize that it attaches to host cells using a trimeric autotransporter adhesin and inhibits their cell wall biosynthesis using a toxin–antitoxin system. Network analyses based on correlating Cand. Roizmanbacterium ADI133 abundances with amplicon sequencing-derived microbial community profiles suggested one potential host organism, classified as a member of the class Thermodesulfovibrionia (Nitrospirae). By providing lactate as an electron donor Cand. Roizmanbacterium ADI133 potentially mediates the transfer of carbon to other microorganisms and thereby is an important connector in the microbial community.     

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A_2A receptor (A_2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A_2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A_2AR C-terminus from 412 to 316 amino acids (A_2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A_2AR levels. As cAMP production is downstream of the first activation event—coupling of G protein to its receptor—investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gα_s proteins, we characterize the association of A_2AR and A_2AΔ316R to Gα_s with and without GDP or GTPγs using surface plasmon resonance (SPR). Gα_s affinity for A_2AR was greatest for apo-Gα_s, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A_2AR C-terminus (A_2AΔ316R) decreased the affinity of the unliganded receptor for Gα_s by ～20%, suggesting small changes to binding can greatly impact downstream signaling.     

Characterization of the PGK2 Associated Microsatellite S0719 on SSC7 Suitable for Parentage and QTL Diagnosis

We isolated and characterized the highly polymorphic tetra-nucleotide microsatellite S0719 on SSC7q14-q15 adjacent to the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene and assigned it to the USDA-MARC linkage map on SSC7 position 77.5 cM closely linked to markers SW859 (76.3 cM) and SWR2036 (79.0 cM). In a panel of 344 individuals representing 11 pig breeds (European, Chinese, and North American), a total of 32 alleles were observed, and the overall breeds' calculated PIC (polymorphism information content), H_E (heterozygosity), and N_E (effective allele number) were 0.94, 0.94, and 16.41. Breed-specific PIC and H_E ranged from 0.66 to 0.87, whereas N_E was as low as 2.95 and as high as 7.96. Considering the high allelic variation of S0719 within and among pig breeds (79% of the genotyped animals were heterozygous), the marker is useful for individual animal identification and parentage determination. Finally, S0719 is also a valuable STS marker for fine-mapping QTL on SSC7 as position 77.5 cM is located in 25 QTL intervals (http://www.animalgenome.org/QTLdb/).     

A Novel Method for the Preparation of Substrate-Free Cytochrome P-45011β

A new method for the removal of the stabilizing substrate, deoxycorticosterone, from adrenal cytochrome P-450_11β, has been developed. Dextran coated charcoal is used for the adsorption of the steroid and the adsorbed steroid is separated from the cytochrome P-450-preparation by low speed centrifugation. The substrate-free enzyme, obtained in this manner, has all the characteristic spectral properties of low-spin cytochrome P-450_11β, and may be converted to the high-spin form by the addition of deoxycorticosterone.

The dextran coated charcoal method has the following advantages over the previously used method of substrate removal. It does not require the addition of the cofactors for cytochrome P-450-dependant hydroxyla-tion of deoxycorticosterone, small amounts of enzyme may be prepared in a short time and the enzyme preparation is not diluted to any great extent during the process.     

Simple and efficient CRISPR/Cas9‐mediated targeted mutagenesis in Xenopus tropicalis

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers. genesis 51:835–843. © 2013 Wiley Periodicals, Inc.