[18F]FEAC and [18F]FEDAC: Two novel positron emission tomography ligands for peripheral-type benzodiazepine receptor in the brain

[¹⁸F]FEAC ([¹⁸F]4a) and [¹⁸F]FEDAC ([¹⁸F]4b) were developed as two novel positron emission tomography (PET) ligands for peripheral-type benzodiazepine receptor (PBR). [¹⁸F]4a and [¹⁸F]4b were synthesized by fluoroethylation of precursors 8a and 8b with [¹⁸F]FCH₂CH₂Br ([¹⁸F]9), respectively. Small-animal PET scan for a neuroinflammatory rat model showed that the two radioligands had high uptakes of radioactivity in the kainic acid-infused striatum, a brain region where PBR density was increased.     

Synthesis of 11C-labeled 2-aminoethanol via a nitroaldol reaction using nitro[11C]methane

The nitroaldol reaction of nitro[¹¹C]methane and formaldehyde using EtOH and EtONa efficiently provided 2-nitro[¹¹C]ethanol in 3 min. The nitro group reduction in the presence of NiCl₂ and NaBH₄ in MeOH followed by purification using semi-preparative HPLC using 10% EtOH aqueous solution as an eluent proved to be a practical and accessible method for the synthesis of 2-amino[2-¹¹C]ethanol.     

CRELD2 is a novel endoplasmic reticulum stress-inducible gene

Recently, endoplasmic reticulum (ER) stress responses have been suggested to play important roles in maintaining various cellular functions and to underlie many tissue dysfunctions. In this study, we first identified cysteine-rich with EGF-like domains 2 (CRELD2) as an ER stress-inducible gene by analyzing a microarray analysis of thapsigargin (Tg)-inducible genes in Neuro2a cells. CRELD2 mRNA is also shown to be immediately induced by treatment with the ER stress-inducing reagents tunicamycin and brefeldin A. In the genomic sequence of the mouse CRELD2 promoter, we found a typical ER stress responsible element (ERSE), which is well conserved among various species. Using a luciferase reporter analyses, we demonstrated that the ERSE in mouse CRELD2 is functional and responds to Tg and ATF6-overexpression. Each mutation of ATF6- or NF-Y-binding sites in the ERSE of the mouse CRELD2 promoter dramatically decreased both the basal activity and responsiveness toward the ER stress stimuli. Our study suggests that CRELD2 could be a novel mediator in regulating the onset and progression of various ER stress-associated diseases.     

Motif WFYY of human PrimPol is crucial to stabilize the incoming 3′-nucleotide during replication fork restart

PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp⁸⁷ and Tyr⁹⁰ residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3′-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.     

Enhanced solubilization of membrane proteins by alkylamines and polyamines

Around 25% of proteins in living organisms are membrane proteins that perform many critical functions such as synthesis of biomolecules and signal transduction. Membrane proteins are extracted from the lipid bilayer and solubilized with a detergent for biochemical characterization; however, their solubilization is an empirical technique and sometimes insufficient quantities of proteins are solubilized in aqueous buffer to allow characterization. We found that addition of alkylamines and polyamines to solubilization buffer containing a detergent enhanced solubilization of membrane proteins from microsomes. The solubilization of polygalacturonic acid synthase localized at the plant Golgi membrane was enhanced by up to 9.9‐fold upon addition of spermidine to the solubilization buffer. These additives also enhanced the solubilization of other plant membrane proteins localized in other organelles such as the endoplasmic reticulum and plasma membrane as well as that of an animal Golgi‐localized membrane protein. Thus, addition of alkylamines and polyamines to solubilization buffer is a generally applicable method for effective solubilization of membrane proteins. The mechanism of the enhancement of solubilization is discussed.     

Fine mapping of <Emphasis Type="Italic">Sta1</Emphasis>, a quantitative trait locus determining stele transversal area,on rice chromosome 9

The stele (root vascular cylinder) in plants plays an important role in the transport of water and nutrients from the root to the shoot. A quantitative trait locus (QTL) on rice chromosome 9 that controls stele transversal area (STA) was previously detected in an F₃ mapping population derived from a cross between the lowland cultivar ‘IR64’, with a small STA, and the upland cultivar ‘Kinandang Patong’, with a large STA. To identify the gene(s) underlying this QTL, we undertook fine mapping of the locus. We screened eight plants from BC₂F₃ lines in which recombination occurred near the QTL. Progeny testing of BC₂F₄ plants was used to determine the genotype classes for the QTL in each BC₂F₃ line. Accordingly, the STA QTL Sta1 (Stele Transversal Area 1) was mapped between the InDel markers ID07_12 and ID07_14. A candidate genomic region for Sta1 was defined more precisely between markers RM566 and RM24334, which delimit a 359-kb interval in the reference cultivar ‘Nipponbare’. A line homozygous for the ‘Kinandang Patong’ allele of Sta1 had an STA approximately 28.4% larger than that of ‘IR64’. However, Sta1 did not influence maximum or total root length, suggesting that this QTL specifically controls STA.     

Relations Between Waist Circumference at Four Sites and Metabolic Risk Factors

The location of waist circumference (WC) measurement differs among diagnostic guidelines for the metabolic syndrome. The present study examined which of four WC measurements was associated most strongly with the clustering of metabolic risk factors in cross‐sectional study. The subjects comprised 1,140 Japanese employees, aged 20–70 years, who underwent health examinations in 2007 and 2008. WC was measured at (i) the narrowest part of the waist, (ii) midway between the lowest rib and the iliac crest, (iii) the umbilical level, and (iv) immediately above the iliac crest. A receiver operator characteristic (ROC) curve was used to assess the ability of each WC measurement to predict the presence of two or more other components of the metabolic syndrome, as defined by the National Cholesterol Education Program's Adult Treatment Panel III (NCEP‐ATP III) in 2005. Multiple risk factors were seen in 43.0% of the men and 12.9% of the women. The minimum and maximum WC measurements differed by 3.9 cm among the men and 12.6 cm among the women. The areas under the curve examining the ability of the four WC measurements to predict the clustering of multiple risk factors were similar. If the same WC cutoff value was applied, the prevalence of the metabolic syndrome changed considerably according to the site of WC measurement. The four WC measurements had similar screening abilities. Given the differences in the WC values according to the site of measurement, WC must be measured at the site specified by each diagnostic guideline.     

Two-dimensional crystallization and analysis of projection images of intact Thermus thermophilus V-ATPase

H(+)-ATPase/synthases are membrane-bound rotary nanomotors that are essential for energy conversion in nearly all life forms. A member of the family of the vacuolar-type ATPases (V-ATPases) from Thermus thermophilus, sometimes also termed A-type ATPase, was purified to homogeneity and subjected to two-dimensional (2D) crystallization trials. A novel approach to the 2D crystallization of unstable complexes yielded densely packed sheets of V-ATPase, exhibiting crystalline arrays. Aggregation of the V-ATPase under acidic conditions during reconstitution circumvented the continuous dissociation of the whole complex into the V(1) and V(o) domains. The resulting three-dimensional aggregates were converted into 2D sheets by the use of a basic buffer, and after a short annealing cycle, ordered arrays of up to 1.5 microm diameter appeared. Fourier transforms calculated from micrographs taken from the negatively stained sample showed diffraction spots to a resolution of 23A. The Fourier transforms of the untilted images revealed unit-cell dimensions of a=232A, b=132A, and gamma=90 degrees , and a projection map was calculated by merging 11 images. The most probable molecular packing suggests p22(1)2(1) symmetry of the crystals and dimer contacts between the V(1) domains.