Potent antagonists of the CCR2b receptor. Part 3: SAR of the (R)-3-aminopyrrolidine series

SAR studies were conducted around lead compound 1 using high-throughput parallel solution and solid phase synthesis. Our lead optimization efforts led to the identification of several CCR2b antagonists with potent activity in both binding and functional assays [Compound 71 CCR2b Binding IC(50) 3.2 nM; MCP-1-Induced Chemotaxis IC(50) 0.83 nM; Ca(2+) Flux IC(50) 7.5 nM].     

Synthesis of FF8181-A

Both enantiomers of FF8181-A were synthesized through optical resolution from the known Diels-Alder reaction product in 15 steps. The absolute configuration of the natural product was determined to be 1S,5S,5aS,9aS,9bS.     

Molecular characterization of organelle-type Nudix hydrolases in Arabidopsis

Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its K(m) value was approximately 100-fold lower for ADP-ribose (13.0+/-0.7 microm) than for ADP-glucose (1,235+/-65 microm). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7+/-3.4 microm) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3+/-5.4 microm) and NADPH (36.9+/-3.5 microm). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1+/-0.9 microm). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n=4-5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes.     

Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by Deep Vent (exo? ) DNA polymerase

In our previous communication we reported the enzymatic recognition of unnatural imidazopyridopyrimidine:naphthyridine (Im:Na) base pairs, i.e. ImO^N:NaN^O and ImN^O:NaO^N, using the Klenow fragment exo⁻ [KF (exo⁻)]. We describe herein the successful results of (i) improved enzymatic recognition for ImN^O:NaO^N base pairs and (ii) further primer extension reactions after the Im:Na base pairs by Deep Vent DNA polymerase exo⁻ [Deep Vent (exo⁻)]. Since KF (exo⁻) did not catalyze primer extension reactions after the Im:Na base pair, we carried out a screening of DNA polymerases to promote the primer extension reaction as well as to improve the selectivity of base pair recognition. As a result, a family B DNA polymerase, especially Deep Vent (exo⁻), seemed most promising for this purpose. In the ImO^N:NaN^O base pair, incorporation of NaN^OTP against ImO^N in the template was preferable to that of the natural dNTPs, while incorporation of dATP as well as dGTP competed with that of ImO^NTP when NaN^O was placed in the template. Thus, the selectivity of base pair recognition by Deep Vent (exo⁻) was less than that by KF (exo⁻) in the case of the ImO^N:NaN^O base pair. On the other hand, incorporation of NaO^NTP against ImN^O in the template and that of ImN^OTP against NaO^N were both quite selective. Thus, the selectivity of base pair recognition was improved by Deep Vent (exo⁻) in the ImN^O:NaO^N base pair. Moreover, this enzyme catalyzed further primer extension reactions after the ImN^O:NaO^N base pair to afford a faithful replicate, which was confirmed by MALDI-TOF mass spectrometry as well as the kinetics data for extension fidelity next to the ImN^O:NaO^N base pair. The results presented in this paper revealed that the ImN^O:NaO^N base pair might be a third base pair beyond the Watson–Crick base pairs.     

Correlation of C4ST-1 and ChGn-2 expression with chondroitin sulfate chain elongation in atherosclerosis

Subendothelial retention of lipoproteins by proteoglycans (PGs) is the initiating event in atherosclerosis. The elongation of chondroitin sulfate (CS) chains is associated with increased low-density lipoprotein (LDL) binding and progression of atherosclerosis. Recently, it has been shown that 2 Golgi enzymes, chondroitin 4-O-sulfotransferase-1 (C4ST-1) and chondroitin N-acetylgalactosaminyltransferase-2 (ChGn-2), play a critical role in CS chain elongation. However, the roles of C4ST-1 and ChGn-2 during the progression of atherosclerosis are not known. The aim of this study was to analyze the expression of C4ST-1 and ChGn-2 in atherosclerotic lesions in vivo and determine whether their expression correlated with CS chain elongation.Low-density lipoprotein receptor knockout (LDLr KO) mice were fed a western diet for 2, 4, and 8 weeks to stimulate development of atherosclerosis. The binding of LDL and CS PG in this mouse model was confirmed by chondroitinase ABC (ChABC) digestion and apolipoprotein B (apo B) staining. Gel filtration analysis revealed that the CS chains began to elongate as early as 2 weeks after beginning a western diet and continued as the atherosclerosis progressed. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) showed that the mRNA levels of C4ST-1 and ChGn-2 increased after 8 weeks of this diet. In contrast, the mRNA levels of their homologs, C4ST-2 and ChGn-1, were unchanged. In addition, immunohistochemical analysis demonstrated that the expression of C4ST-1 and ChGn-2 appeared to have similar site-specific patterns and coincided with biglycan expression at the aortic root.Our results suggested that C4ST-1 and ChGn-2 may be involved in the elongation of CS chains in the arterial wall during the progression of atherosclerosis. Therefore, modulating their expression and activity might be a novel therapeutic strategy for atherosclerosis.     

Effects of conformational peptide probe DP4 on bidirectional signaling between DHPR and RyR1 calcium channels in voltage-clamped skeletal muscle fibers

In skeletal muscle, excitation-contraction coupling involves the activation of dihydropyridine receptors (DHPR) and type-1 ryanodine receptors (RyR1) to produce depolarization-dependent sarcoplasmic reticulum Ca²⁺ release via orthograde signaling. Another form of DHPR-RyR1 communication is retrograde signaling, in which RyRs modulate the gating of DHPR. DP4 (domain peptide 4), is a peptide corresponding to residues Leu²⁴⁴²-Pro²⁴⁷⁷ of the central domain of the RyR1 that produces RyR1 channel destabilization. Here we explore the effects of DP4 on orthograde excitation-contraction coupling and retrograde RyR1-DHPR signaling in isolated murine muscle fibers. Intracellular dialysis of DP4 increased the peak amplitude of Ca²⁺ release during step depolarizations by 64% without affecting its voltage-dependence or kinetics, and also caused a similar increase in Ca²⁺ release during an action potential waveform. DP4 did not modify either the amplitude or the voltage-dependence of the intramembrane charge movement. However, DP4 augmented DHPR Ca²⁺ current density without affecting its voltage-dependence. Our results demonstrate that the conformational changes induced by DP4 regulate both orthograde E-C coupling and retrograde RyR1-DHPR signaling.     

SOD1 (copper/zinc superoxide dismutase) deficiency drives amyloid β protein oligomerization and memory loss in mouse model of Alzheimer disease

Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid β (Aβ) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282-11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aβ oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of N(ε)-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.     

Klotho inhibits transforming growth factor-beta1 (TGF-beta1) signaling and suppresses renal fibrosis and cancer metastasis in mice

Fibrosis is a pathological process characterized by infiltration and proliferation of mesenchymal cells in interstitial space. A substantial portion of these cells is derived from residing non-epithelial and/or epithelial cells that have acquired the ability to migrate and proliferate. The mesenchymal transition is also observed in cancer cells to confer the ability to metastasize. Here, we show that renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts are suppressed by administration of secreted Klotho protein to mice. Klotho is a single-pass transmembrane protein expressed in renal tubular epithelial cells. The extracellular domain of Klotho is secreted by ectodomain shedding. Secreted Klotho protein directly binds to the type-II TGF-β receptor and inhibits TGF-β1 binding to cell surface receptors, thereby inhibiting TGF-β1 signaling. Klotho suppresses TGF-β1-induced epithelial-to-mesenchymal transition (EMT) responses in cultured cells, including decreased epithelial marker expression, increased mesenchymal marker expression, and/or increased cell migration. In addition to TGF-β1 signaling, secreted Klotho has been shown to inhibit Wnt and IGF-1 signaling that can promote EMT. These results have raised the possibility that secreted Klotho may function as an endogenous anti-EMT factor by inhibiting multiple growth factor signaling pathways simultaneously.     

Two species of Strobilomyces (Boletaceae, Boletales), S. seminudus and S. hongoi sp. nov. from Japan

We describe and illustrate two Strobilomyces species, S. seminudus and S. hongoi sp. nov. These two species have been confused and treated as a single species (i.e. S. seminudus). However recent studies based on population genetics have implied that they are reproductively isolated. In the present study we found that they are phylogenetically and morphologically distinct. The molecular phylogenetic trees inferred from the partial sequences of the largest subunit of RNA polymerase II (RPB1) and the second-largest subunit of RNA polymerase II (RPB2) support the differentiation of these two species as well as their differentiation from other related species. Strobilomyces seminudus is characterized by a stipe with an annular zone, becoming distinctly thickened near the apex and mottled with appressed-tomentose scales near the base. In contrast S. hongoi is characterized by a stipe with a remarkable reticulum at the upper and middle part and with minutely warty scales downward. Stipe characteristics also are useful for distinguishing these two species from other related species. In addition the incompletely reticulated basidiospores of these two species are also distinct from those of related species (i.e. S. foveatus).     

Amplification of a plasmid bearing a mammalian replication initiation region in chromosomal and extrachromosomal contexts

Amplified genes in cancer cells reside on extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSRs). We used a plasmid bearing a mammalian replication initiation region to model gene amplification. Recombination junctions in the amplified region were comprehensively identified and sequenced. The junctions consisted of truncated direct repeats (type 1) or inverted repeats (type 2) with or without spacing. All of these junctions were frequently detected in HSRs, whereas there were few type 1 or a unique type 2 flanked by a short inverted repeat in DMs. The junction sequences suggested a model in which the inverted repeats were generated by sister chromatid fusion. We were consistently able to detect anaphase chromatin bridges connected by the plasmid repeat, which were severed in the middle during mitosis. De novo HSR generation was observed in live cells, and each HSR was lengthened more rapidly than expected from the classical breakage/fusion/bridge model. Importantly, we found massive DNA synthesis at the broken anaphase bridge during the G1 to S phase, which could explain the rapid lengthening of the HSR. This mechanism may not operate in acentric DMs, where most of the junctions are eliminated and only those junctions produced through stable intermediates remain.