Symmetrical approach of spiro-pyrazolidinediones as acetyl-CoA carboxylase inhibitors

Spiro-pyrazolidinedione derivatives without quaternary chiral center were discovered by structure-based drug design and characterized as potent acetyl-CoA carboxylase (ACC) inhibitors. The high metabolic stability of the spiro-pyrazolo[1,2-a]pyridazine scaffold and enhancement of the activity by incorporation of a 7-methoxy group on the benzothiophene core successfully led to the identification of compound 4c as an orally bioavailable and highly potent ACC inhibitor. Oral administration of 4c significantly decreased the values of the respiratory quotient in rats, indicating the stimulation of fatty acid oxidation.     

Pyrrolidinyl phenylurea derivatives as novel CCR3 antagonists

Optimization starting with our lead compound 1 (IC₅₀ = 4.9 nM) led to the identification of pyrrolidinyl phenylurea derivatives. Further modification toward improvement of the bioavailability provided (R)-1-(1-((6-fluoronaphthalen-2-yl)methyl)pyrrolidin-3-yl)-3-(2-(2-hydroxyethoxy)phenyl)urea 32 (IC₅₀ = 1.7 nM), a potent and orally active CCR3 antagonist.     

Evidence of the existence of a toxic form of Cylindrospermopsis raciborskii (Nostocales,Cyanobacteria) in Japan

Cylindrospermopsis raciborskii is a common, bloom‐forming, planktonic, freshwater cyanobacterium. Toxic populations producing cylindrospermopsin can cause water‐safety problems. Although C. raciborskii is distributed worldwide, the presence of cylindrospermopsin‐producing strains of C. raciborskii was initially reported only in Australia and recently in Thailand. Here, we report the isolation of a toxic strain of C. raciborskii (ISG9) from a freshwater sample collected in Okinawa in 2008. This is the first report describing toxin expression in this species in Japan, detected from a subtropical area. The C. raciborskii species is known to produce cylindrospermopsin as a dominant toxin; however, in this new isolate, the dominant toxin expressed was deoxy‐cylindrospermopsin. The discovery of a toxic strain of C. raciborskii in southern Japan emphasizes the need for basic monitoring schemes for this species in water supplies located in the temperate regions of Japan because of its possible expansion and distribution to other geographic areas.     

Large-scale development of expressed sequence tag-derived simple sequence repeat markers and diversity analysis in Arachis spp.

Large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed in peanut (Arachis hypogaea L.) to obtain more informative genetic markers. A total of 10,102 potential non-redundant EST sequences, including 3,445 contigs and 6,657 singletons, were generated from cDNA libraries of the gynophore, roots, leaves and seedlings. A total of 3,187 primer pairs were designed on flanking regions of SSRs, some of which allowed one and two base mismatches. Among the 3,187 markers generated, 2,540 (80%) were trinucleotide repeats, 302 (9%) were dinucleotide repeats, and 345 (11%) were tetranucleotide repeats. Pre-polymorphic analyses of 24 Arachis accessions were performed using 10% polyacrylamide gels. A total of 1,571 EST-SSR markers showing clear polymorphisms were selected for further polymorphic analysis with a Fluoro-fragment Analyzer. The 16 Arachis accessions examined included cultivated peanut varieties as well as diploid species with the A or B genome. Altogether 1,281 (81.5%) of the 1,571 markers were polymorphic among the 16 accessions, and 366 (23.3%) were polymorphic among the 12 cultivated varieties. Diversity analysis was performed and the genotypes of all 16 Arachis accessions showed similarity coefficients ranging from 0.37 to 0.97. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9604-8) contains supplementary material, which is available to authorized users.     

Indirect effects of Wnt3a/β-catenin signalling support mouse spermatogonial stem cells in vitro

Proper regulation of spermatogonial stem cells (SSCs) is crucial for sustaining steady-state spermatogenesis. Previous work has identified several paracrine factors involved in this regulation, in particular, glial cell line-derived neurotrophic factor and fibroblast growth factor 2, which promote long-term SSC self-renewal. Using a SSC culture system, we have recently reported that Wnt5a promotes SSC self-renewal through a β-catenin-independent Wnt mechanism whereas the β-catenin-dependent Wnt pathway is not active in SSCs. In contrast, another study has reported that Wnt3a promotes SSC self-renewal through the β-catenin-dependent pathway, as it can stimulate the proliferation of a spermatogonia cell line. To reconcile these two contradictory reports, we assessed Wnt3a effects on SSCs and progenitor cells, rather than a cell line, in vitro. We observed that Wnt3a induced β-catenin-dependent signalling in a large subset of germ cells and increased SSC numbers. However, further investigation revealed that cell populations with greater β-catenin-signalling activity contained fewer SSCs. The increased maintenance of SSCs by Wnt3a coincided with more active cell cycling and the formation of germ cell aggregates, or communities, under feeder-free conditions. Therefore, the results of this study suggest that Wnt3a selectively stimulates proliferation of progenitors that are committed to differentiation or are in the process of exiting the SSC state, leading to enhanced formation of germ cell communities, which indirectly support SSCs and act as an in vitro niche.     

Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor for HLA-C than KIR2DL3

Interactions between HLA-C ligands and inhibitory killer cell Ig-like receptors (KIR) control the development and response of human NK cells. This regulatory mechanism is usually described by mutually exclusive interactions of KIR2DL1 with C2 having lysine 80, and KIR2DL2/3 with C1 having asparagine 80. Consistent with this simple rule, we found from functional analysis and binding assays to 93 HLA-A, HLA-B, and HLA-C isoforms that KIR2DL1*003 bound all C2, and only C2, allotypes. The allotypically related KIR2DL2*001 and KIR2DL3*001 interacted with all C1, but they violated the simple rule through interactions with several C2 allotypes, notably Cw*0501 and Cw*0202, and two HLA-B allotypes (B*4601 and B*7301) that share polymorphisms with HLA-C. Although the specificities of the "cross-reactions" were similar for KIR2DL2*001 and KIR2DL3*001, they were stronger for KIR2DL2*001, as were the reactions with C1. Mutagenesis explored the avidity difference between KIR2DL2*001 and KIR2DL3*001. Recombinant mutants mapped the difference to the Ig-like domains, where site-directed mutagenesis showed that the combination, but not the individual substitutions, of arginine for proline 16 in D1 and cysteine for arginine 148 in D2 made KIR2DL2*001 a stronger receptor than KIR2DL3*001. Neither residue 16 or 148 is part of, or near to, the ligand-binding site. Instead, their juxtaposition near the flexible hinge between D1 and D2 suggests that their polymorphisms affect the ligand-binding site by changing the hinge angle and the relative orientation of the two domains. This study demonstrates how allelic polymorphism at sites distal to the ligand-binding site of KIR2DL2/3 has diversified this receptor's interactions with HLA-C.     

Expression and localization of aquaporins in the kidney of the musk shrew (Suncus murinus).

Expression and localization of members of the aquaporin (AQP) family (AQP1, 2, 3, 4, and 5) in the kidney of the musk shrew (Suncus murinus) was examined by immunohistochemistry. AQP1 was expressed in the proximal tubules and in the thin limb of the loops of Henle. AQP1 was the only water channel expressed in the proximal nephron examined, indicating that AQP1 may be an independent water transporter in the proximal nephron. AQP2 and AQP5 were localized to the apical cytoplasm of the cortical to medullary collecting duct (CD) cells and AQP3 and AQP4 were localized to the basal aspect of the cortical to medullary CD cells. AQP3 expression was weaker in the cortical cells compared with the medullary cells, whereas AQP4 was strongly positive throughout the CD. These indicate that the CD is the main water reabsorption segment of the nephron and is regulated by AQPs. Indeed, apical water transport of CD cells of the musk shrew may be controlled by both AQP2 and AQP5. The characteristic expression pattern of the AQPs in this animal provides a novel animal model for elucidating the regulation of water reabsorption by AQPs in the mammalian kidney.     

Design and synthesis of versatile ganglioside probes for carbohydrate microarrays

A series of ganglioside GM1-, GM2-, and GM3-type probes, in which the ceramide portion is replaced with a glucose residue, were systematically synthesized based on a convergent synthetic method.