In Vivo Differences between Two Optical Isomers of Radioiodinated o-iodo-trans-decalinvesamicol for Use as a Radioligand for the Vesicular Acetylcholine Transporter

Purpose

To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.

Procedures

Radioiodinated o-iodo-trans-decalinvesamicol ([¹²⁵I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [¹²⁵I]OIDV was separated into its two optical isomers (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [¹²⁵I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [¹²⁵I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [¹²⁵I]OIDV isomers and (-)-[¹²³I]OIDV for SPECT at 60 min postinjection.

Results

VAChT binding affinity (Ki) of (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[¹²⁵I]OIDV was the same as that of (+)-[¹²⁵I]OIDV. However, (+)-[¹²⁵I]OIDV clearance from the brain was faster than (-)-[¹²⁵I]OIDV. At 30 min post-injection, accumulation of (-)-[¹²⁵I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[¹²⁵I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[¹²⁵I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[¹²⁵I]OIDV in these regions. Furthermore, (-)-[¹²³I]OIDV for SPECT showed the same regional brain distribution as (-)-[¹²⁵I]OIDV.

Conclusion

These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain.     

A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature

DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold.     

Partial contribution of mitochondrial permeability transition to t-butyl hydroperoxide-induced cell death

Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 μM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress.     

Sarcoplasmic Reticulum Structure and Functional Properties that Promote Long-Lasting Calcium Sparks

Calcium (Ca) sparks are the fundamental sarcoplasmic reticulum (SR) Ca release events in cardiac myocytes, and they have a typical duration of 20–40 ms. However, when a fraction of ryanodine receptors (RyRs) are blocked by tetracaine or ruthenium red, Ca sparks lasting hundreds of milliseconds have been observed experimentally. The fundamental mechanism underlying these extremely prolonged Ca sparks is not understood. In this study, we use a physiologically detailed mathematical model of subcellular Ca cycling to examine how Ca spark duration is influenced by the number of functional RyRs in a junctional cluster (which is reduced by tetracaine or ruthenium red) and other SR Ca handling properties. One RyR cluster contains a few to several hundred RyRs, and we use a four-state Markov RyR gating model. Each RyR opens stochastically and is regulated by cytosolic and luminal Ca. We varied the number of functional RyRs in the single cluster, diffusion within the SR network, diffusion between network and junctional SR, cytosolic Ca diffusion, SERCA uptake activity, and RyR open probability. For long-lasting Ca release events, opening events within the cluster must occur continuously because the typical open time of the RyR is only a few milliseconds. We found the following: 1) if the number of RyRs is too small, it is difficult to maintain consecutive openings and stochastic attrition terminates the release; 2) if the number of RyRs is too large, the depletion of Ca from the junctional SR terminates the release; and 3) very long release events require relatively small-sized RyR clusters (reducing flux as seen experimentally with tetracaine) and sufficiently rapid intra-SR Ca diffusion, such that local junctional intra-SR [Ca] can be maintained by intra-SR diffusion and overall SR Ca reuptake.     

Decrease in Hydraulic Conductivity of a Paddy Field using Biocalcification in situ

The hydraulic conductivity of a paddy field (Anthraquic Dystrustept), a silty clay soil containing more than 29% (w/w) of gravel, in Nagoya University Farm was reduced by in situ treatment of subsurface soil using bentonite and biocalcification (microbial calcium carbonate precipitation) through the addition of CaCl₂, urea, and corn steep liquor (CSL). The treatment decreased the hydraulic conductivity of the field from an average of 10^?3 cm/s to a range of 10^?5 to 10^?7 cm/s during 69 days, with reducing the proportion of pores of subsurface soil larger than 75 µm in diameter. The biocalcification effect was observed at 10-cm thickness from the treated subsurface. Laboratory soil core experiments demonstrated that the decrease in the hydraulic conductivity was not attributed to the effect of bentonite but mainly to the effect of biocalcification. The addition of CSL enhanced the urease activity of soil required for biocalcification, even at 4°C, as indicated by a decrease in urease activation energy temperature sensitivity. These experimental results agreed with the gradual decrease in hydraulic conductivity observed in the field when the average daily temperature was 7°C (days 24–69). It was suggested that the biocalcification is a potential technique to reduce the hydraulic conductivity of paddy field.     

Macroinvertebrate assemblages in an artificial habitat—a settling basin of a hydroelectric power plant: comparison with a natural riffle habitat

The macroinvertebrate assemblages in an artificial habitat, the settling basin of a hydroelectric power plant, were investigated and compared with those in a natural habitat, riffles in a nearby river. This study showed that macroinvertebrate density was much higher in the settling basin than in the riffles. Macroinvertebrate assemblage composition differed between the settling basin and the riffles. The difference was probably due to the widespread bryophyte beds in the settling basin. Cincticostella, Brachycentrus, Ephmerella, and chironomid midges, which are usually abundant in bryophyte beds, were present at much higher densities in the settling basin. Cheumatopsyche and Taeniopterygidae were also present at higher densities in the settling basin than in the natural riffles. In contrast, Epeorus was present at lower density in the settling basin than in the natural riffles. This study suggests that the settling basin increases β-diversity in riverine ecosystems.     

Structure-activity relationship studies of Bz amide-containing α-GalCer derivatives as natural killer T cell modulators

Abstract

CD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d.     

CTENO64 Is Required for Coordinated Paddling of Ciliary Comb Plate in Ctenophores

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Optimization of 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines to generate a highly selective PI3Kδ inhibitor

Chemical optimization of the 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine (THPP) scaffold was conducted with a focus on cellular potency while maintaining high selectivity against PI3K isoforms. Compound 11f was identified as a potent, highly selective and orally available PI3Kδ inhibitor. In addition, 11f exhibited efficacy in an in vivo antibody production model. The desirable drug-like properties and in vivo efficacy of 11f suggest its potential as a drug candidate for the treatment of autoimmune diseases and leukocyte malignancies.