3D QSAR study, synthesis, and in vitro evaluation of (+)-5-FBVM as potential PET radioligand for the vesicular acetylcholine transporter (VAChT).

Located in presynaptic cholinergic nerve terminals, the vesicular acetylcholine transporter (VAChT) represents a potential target for quantitative visualization of early degeneration of cholinergic neurons in Alzheimer's disease using PET. Benzovesamicol derivatives are proposed as radioligands for this purpose. We report QSAR studies of vesamicol and benzovesamicol derivatives taking into account the stereoselectivity of the VAChT binding site. Use of different data sets and different models in this study revealed that both enantiomers of 5-fluoro-3-(4-phenyl-piperidin-1-yl)-1,2,3,4-tetrahydro-naphthalen-2-ol (5-FBVM) are promising candidates, with predicted VAChT affinities between 6.1 and 0.05 nM. The synthesis of enantiopure (R,R)- and (S,S)-5-FBVM and their corresponding triazene precursors for future radiofluorination is reported. Both enantiomers exhibited high in vitro affinity for VAChT [(+)-5-FBVM: K(i)=6.95 nM and (-)-5-FBVM: K(i)=3.68 nM] and were selective for σ(2) receptors (∼70-fold), only (+)-5-FBVM is selective for σ(1) receptors (∼fivefold). These initial results suggest that (+)-(S,S)-5-FBVM warrants further investigation as a potential radioligand for in vivo PET imaging of cholinergic nerve terminals.

Phylogenetic diversity and metagenomics of candidate division OP3.

Except for environmental 16S rRNA gene sequences, no information is available for members of the candidate division OP3. These bacteria appear to thrive in anoxic environments, such as marine sediments, hypersaline deep sea, freshwater lakes, aquifers, flooded paddy soils and methanogenic bioreactors. The 16S rRNA phylogeny suggests that OP3 belongs to the Planctomycetes/Verrucomicrobia/Chlamydiae (PVC) superphylum. Metagenomic fosmid libraries were constructed from flooded paddy soil and screened for 16S rRNA gene-containing fragments affiliated with the PVC superphylum. The screening of 63 000 clones resulted in 23 assay-positive fosmids, of which three clones were affiliated with OP3. The 16S rRNA gene sequence divergence between the fragments OP3/1, OP3/2 and OP3/3 ranges from 18% to 25%, indicating that they belong to different OP3 subdivisions. The 23S rRNA phylogeny confirmed the membership of OP3 in the PVC superphylum. Sequencing the OP3 fragments resulted in a total of 105 kb of genomic information and 90 ORFs, of which 47 could be assigned a putative function and 11 were conserved hypothetical. Using BLASTP searches, a high proportion of ORFs had best matches to homologues from Deltaproteobacteria, rather than to those of members of the PVC superphylum. On the fragment OP3/3, a cluster of nine ORFs was predicted to encode the bacterial NADH dehydrogenase I. Given the high proportion of homologues present in deltaproteobacteria and anoxic conditions in the natural environment of OP3 bacteria, the detection of NADH dehydrogenase I may suggest an anaerobic respiration mode. Oligonucleotide frequencies calculated for OP3/1, OP3/2 and OP/3 show high intraphylum correlations. This novel sequence information could therefore be used to identify OP3-related fragments in large metagenomic data sets using marker gene-independent procedures in the future. In addition to the OP3 fragments, a single metagenomic fragment affiliated with the candidate division BRC1 was obtained and analysed.

Bavariicoccus seileri gen. nov., sp. nov., isolated from the surface and smear water of German red smear soft cheese.

The phylogenetic position and physiological characters of six hitherto-unknown lactic acid bacterial isolates, which form part of the surface microbiota of German red smear soft cheese, are reported. The coccoid cells are aerotolerant, Gram-positive, catalase-negative and non-motile. The cell-wall peptidoglycan contains alanine, glutamic acid, lysine and aspartic acid and is of the A4alpha type (l-Lys-d-Asp). The sequences of the 16S rRNA, groEL and rpoB genes of the six isolates are identical and reveal that these isolates represent an independent lineage within the radiation of the family Enterococcaceae in the phylum Firmicutes. Their closest phylogenetic neighbour is the lactic acid bacterium Atopobacter phocae M1590/94/2(T), with which they share 94.9 % 16S rRNA gene sequence similarity; representatives of other genera such as Granulicatella, Carnobacterium and Trichococcus are more distantly related. DNA-DNA hybridization studies reveal that the six isolates are members of a single species, and this is confirmed by similarities in biochemical characteristics. The six isolates were assigned four different groups by Fourier-transform infrared and randomly amplified polymorphic DNA typing. Therefore, it is formally proposed that these isolates should be classified in a single novel species of a novel genus and be named Bavariicoccus seileri gen. nov., sp. nov. The type strain of Bavariicoccus seileri is WCC 4188(T) (=DSM 19936(T) =CCUG 55508(T)).