Synthesis of novel benzbromarone derivatives designed to avoid metabolic activation

We synthesized six novel BBR derivatives that were designed to avoid metabolic activation via ipso-substitution and evaluated for their degree of toxicity and hURAT1 inhibition. It was found that all of the derivatives demonstrate lower cytotoxicity in mouse hepatocytes and lower levels of metabolic activation than BBR, while maintaining their inhibitory activity toward the uric acid transporter. We propose that these derivatives could serve as effective uricosuric agents that have much better safety profiles than BBR.     

A simple and efficient synthesis of puromycin, 2,2′-anhydro-pyrimidine nucleosides,cytidines and 2′,3′-anhydroadenosine from 3′,5′-O-sulfinyl Xylo-nucleosides

Synthesis of antibiotics, puromycin and 3 ′-amino-3 ′-deoxy-N ⁶,N ⁶-dimethyladenosine 11 was achieved by utilizing the cyclic sulfite 6a of the xylo-3 ′,5 ′-dihydroxy group as a new protective group. The key synthetic step is the deprotection of the sulfite moiety through the intramolecular cyclization of 2-α-carbamate 7. In a similar manner, 2,2 ′-anhydro-pyrimidine nucleosides 15, ribo-cytidines 17 and 2 ′,3 ′-anhydroadenosine 14 were prepared in high yields from the corresponding sulfites 4, 5, and 6b, respectively.     

The assembly of the FtsZ ring at the mid-chloroplast division site depends on a balance between the activities of AtMinE1 and ARC11/AtMinD1

Chloroplast division comprises a sequence of events that facilitatesymmetric binary fission and that involve prokaryotic-like stromaldivision factors such as tubulin-like GTPase FtsZ and the divisionsite regulator MinD. In Arabidopsis, a nuclear-encoded prokaryoticMinE homolog, AtMinE1, has been characterized in terms of itseffects on a dividing or terminal chloroplast state in a limitedseries of leaf tissues. However, the relationship between AtMinE1expression and chloroplast phenotype remains to be fully elucidated.Here, we demonstrate that a T-DNA insertion mutation in AtMinE1results in a severe inhibition of chloroplast division, producingmotile dots and short filaments of FtsZ. In AtMinE1 sense (overexpressor)plants, dividing chloroplasts possess either single or multipleFtsZ rings located at random intervals and showing constrictiondepth, mainly along the chloroplast polarity axis. The AtMinE1sense plants displayed equivalent chloroplast phenotypes toarc11, a loss-of-function mutant of AtMinD1 which forms replicatingmini-chloroplasts. Furthermore, a certain population of FtsZrings formed within developing chloroplasts failed to initiateor progress the membrane constriction of chloroplasts and consequentiallyto complete chloroplast fission in both AtMinE1 sense and arc11/atminD1plants. Our present data thus demonstrate that the chloroplastdivision site placement involves a balance between the opposingactivities of AtMinE1 and AtMinD1, which acts to prevent FtsZring formation anywhere outside of the mid-chloroplast. In addition,the imbalance caused by an AtMinE1 dominance causes multiple,non-synchronous division events at the single chloroplast level,as well as division arrest, which becomes apparent as the chloroplastsmature, in spite of the presence of FtsZ rings.     

Inhibitory synaptic transmission tuned by Ca2+ and glutamate through the control of GABAAR lateral diffusion dynamics

The GABAergic synapses, a primary inhibitory synapse in the mammalian brain, is important for the normal development of brain circuits, and for the regulation of the excitation-inhibition balance critical for brain function from the developmental stage throughout life. However, the molecular mechanism underlying the formation, maintenance, and modulation of GABAergic synapses is less understood compared to that of excitatory synapses. Quantum dot-single particle tracking (QD-SPT), a super-resolution imaging technique that enables the analysis of membrane molecule dynamics at single-molecule resolution, is a powerful tool to analyze the behavior of proteins and lipids on the plasma membrane. In this review, we summarize the recent application of QD-SPT in understanding of GABAergic synaptic transmission. Here we introduce QD-SPT experiments that provide further insights into the molecular mechanism supporting GABAergic synapses. QD-SPT studies revealed that glutamate and Ca²⁺ signaling is involved in (a) the maintenance of GABAergic synapses, (b) GABAergic long-term depression, and GABAergic long-term potentiation, by specifically activating signaling pathways unique to each phenomenon. We also introduce a novel Ca²⁺ imaging technique to describe the diversity of Ca²⁺ signals that may activate the downstream signaling pathways that induce specific biological output.     

Calcium-activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca²⁺ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.     

Enhancement of Stimulation-Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin

Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 microM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 microM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mM K+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.     

Targeting Photoreceptors via Intravitreal Delivery Using Novel,Capsid-Mutated AAV Vectors

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.     

Analysis of sialyltransferase-like proteins from Oryza sativa

Sialic acids are widely distributed among living creatures, from bacteria to mammals, but it has been commonly accepted that they do not exist in plants. However, with the progress of genome analyses, putative gene homologs of animal sialyltransferases have been detected in the genome of some plants. In this study, we cloned three genes from Oryza sativa (Japanese rice) that encode sialyltransferase-like proteins, designated OsSTLP1, 2, and 3, and analyzed the enzymatic activity of the proteins. OsSTLP1, 2, and 3 consist of 393, 396, and 384 amino acids, respectively, and each contains sequences similar to the sialyl motifs that are highly conserved among animal sialyltransferases. The recombinant soluble forms of OsSTLPs produced by COS-7 cells were analyzed for sialyltransferase-like activity. OsSTLP1 exhibited such activity toward the oligosaccharide Galbeta1,4GlcNAc and such glycoproteins as asialofetuin, alpha1-acid glycoprotein, and asialo-alpha1-acid glycoprotein; OsSTLP3 exhibited similar activity toward asialofetuin; and OsSTLP2 exhibited no sialyltransferase-like activity. The sialic acid transferred by OsSTLP1 or 3 was linked to galactose of Galbeta1,4GlcNAc through alpha2,6-linkage. This is the first report of plant proteins having sialyltransferase-like activity.