Design and synthesis of a series of α-benzyl phenylpropanoic acid-type peroxisome proliferator-activated receptor (PPAR) gamma partial agonists with improved aqueous solubility

In the continuing study directed toward the development of peroxisome proliferator-activated receptor gamma (hPPARγ) agonist, we attempted to improve the water solubility of our previously developed hPPARγ-selective agonist 3, which is insufficiently soluble for practical use, by employing two strategies: introducing substituents to reduce its molecular planarity and decreasing its hydrophobicity via replacement of the adamantyl group with a heteroaromatic ring. The first approach proved ineffective, but the second was productive. Here, we report the design and synthesis of a series of α-benzyl phenylpropanoic acid-type hPPARγ partial agonists with improved aqueous solubility. Among them, we selected (R)-7j, which activates hPPARγ to the extent of about 65% of the maximum observed with a full agonist, for further evaluation. The ligand-binding mode and the reason for the partial-agonistic activity are discussed based on X-ray-determined structure of the complex of hPPARγ ligand-binding domain (LBD) and (R)-7j with previously reported ligand-LDB structures. Preliminal apoptotic effect of (R)-7j against human scirrhous gastric cancer cell line OCUM-2MD3 is also described.     

Localization of 9- and 13-oxo-octadecadienoic acids in tomato fruit

We previously reported that the two peroxisome proliferator-activated receptor-α agonists, 9- and 13-oxo-octadecadienoic acids (oxo-ODAs), were found in the tomato fruit. However, their localization remains unknown. Herein, we showed that oxo-ODAs localize primarily in the fruit peel and their amount increases after the homogenization of the tomato fruit.     

Nematocidal activity of 6-O-octanoyl- and 6-O-octyl-d-allose against larvae of Caenorhabditis elegans

ABSTRACT

The nematocidal activities of the fatty acid esters of d-allose were examined using the larvae of C. elegans. Among the fatty acid esters, 6-O-octanoyl-d-allose (3) showed significant activity. 6-O-octanoyl-d-glucose (5) showed no activity, indicating that the D-allose moiety is essential for the nematocidal activity of 3. A nonhydrolyzable alkoxy analog 6-O-octyl-d-allose (6) also showed activity equivalent to that of 3.     

Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids

The ¹H–¹³C HMQC signals of the ¹³CH₃ moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ₁-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically ¹³CH₃-labeled [U–²H;¹⁵N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G.     

Novel mutations of CDKN1C in Japanese patients with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an imprinting-related human disease that is characterized by macrosomia, macroglossia, abdominal wall defects, and variable minor features. BWS is caused by several genetic/epigenetic alterations, such as loss of methylation at KvDMR1, gain of methylation at H19-DMR, paternal uniparental disomy of chromosome 11, CDKN1C mutations, and structural abnormalities of chromosome 11. CDKN1C is an imprinted gene with maternal preferential expression, encoding for a cyclin-dependent kinase (CDK) inhibitor. Mutations in CDKN1C are found in 40 % of familial BWS cases with dominant maternal transmission and in ~5 % of sporadic cases. In this study, we searched for CDKN1C mutations in 37 BWS cases that had no evidence for other alterations. We found five mutations—four novel and one known—from a total of six patients. Four were maternally inherited and one was a de novo mutation. Two frame-shift mutations and one nonsense mutation abolished the QT domain, containing a PCNA-binding domain and a nuclear localization signal. Two missense mutations occurred in the CDK inhibitory domain, diminishing its inhibitory function. The above-mentioned mutations were predicted by in silico analysis to lead to loss of function; therefore, we strongly suspect that such anomalies are causative in the etiology of BWS.     

Inferences of evolutionary history of a widely distributed mangrove species,Bruguiera gymnorrhiza,in the Indo‐West Pacific region

Inference of genetic structure and demographic history is fundamental issue in evolutionary biology. We examined the levels and patterns of genetic variation of a widespread mangrove species in the Indo‐West Pacific region, Bruguiera gymnorrhiza, using ten nuclear gene regions. Genetic variation of individual populations covering its distribution range was low, but as the entire species it was comparable to other plant species. Genetic differentiation among the investigated populations was high. They could be divided into two genetic clusters: the West and East clusters of the Malay Peninsula. Our results indicated that these two genetic clusters derived from their ancestral population whose effective size of which was much larger compared to the two extant clusters. The point estimate of speciation time between B. gymnorrhiza and Bruguiera sexangula was two times older than that of divergence time between the two clusters. Migration from the West cluster to the East cluster was much higher than the opposite direction but both estimated migration rates were low. The past Sundaland and/or the present Malay Peninsula are likely to prevent gene flow between the West and East clusters and function as a geographical or land barrier.     

TGF-?1 Enhances the BMP-2-Induced Chondrogenesis of Bovine Synovial Explants and Arrests Downstream Differentiation at an Early Stage of Hypertrophy

Background

Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2) induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2) and transforming growth factor beta 1 (TGF-ß1) were investigated.

Methodology/Principal Findings

Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml) for 4 (or 6) weeks. FGF-2 (10 ng/ml) or TGF-ß1 (10 ng/ml) was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs) only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2), but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume.

Conclusions/Significance

TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of clinical repair, our findings will be of importance in fine-tuning the stimulation protocol for the optimal chondrogenic differentiation of synovial explants.     

Association between Soluble (Pro)Renin Receptor Concentration in Cord Blood and Small for Gestational Age Birth: A Cross-Sectional Study

Objective

The (pro)renin receptor [(P)RR] has been recognized as a multifunctional receptor. The purpose of this study was to assess the association between plasma soluble (P)RR [s(P)RR] concentration in human cord blood (i.e., neonatal blood at birth) and small for gestational age (SGA) birth.

Methods

Participants were women with a singleton pregnancy who delivered at the National Center for Child Health and Development between January 2010 and December 2011. Inclusion criteria were availability of maternal pre-pregnancy and paternal body mass index, and the absence of structural anomalies in neonates. s(P)RR concentration in cord blood was measured in 621 neonates. The 621 pairs of mothers and neonates were categorized into four groups based on quartiles of s(P)RR concentrations in cord blood. SGA was defined as a birth weight below the 10^th percentile for gestational age. Logistic regression analysis was performed to assess the association between cord plasma s(P)RR concentration (quartiles) and incidence of SGA births.

Results

Among 621 neonates, 55 (8.9%) were diagnosed as SGA (SGA group) and 566 (91.1%) were not (non-SGA group). Average s(P)RR concentration in cord blood was 66.1±12.6 ng/ml (mean±standard deviation). There were 155 pairs in the first plasma s(P)RR concentration quartile (Q1: <58.2 ng/ml), 153 pairs in the second quartile (Q2: 58.2–65.1 ng/ml), 157 pairs in the third quartile (Q3: 65.1–73.1 ng/ml) and 156 pairs in the fourth quartile (Q4: >73.1 ng/ml). The distribution of SGA births was 18 (11.6%) in Q1, 14 (9.2%) in Q2, 16 (10.2%) in Q3 and 7 (4.5%) in Q4, respectively. The odds ratio of SGA births was 0.24 (95% confidence interval: 0.08–0.71) for the fourth quartile compared to the first quartile in multivariate models. The P-value for trend was also significant (P = 0.020).

Conclusion

High s(P)RR concentration is associated with a lower SGA birth likelihood.     

Immunotherapeutic targeting of LIGHT/LTβR/HVEM pathway fully recapitulates the reduced cytotoxic phenotype of LIGHT-deficient T cells

Tumor necrosis factor (TNF)/TNF receptor (TNFR) superfamily members play essential roles in the development of the different phases of the immune response. Mouse LIGHT (TNFSF14) is a type II transmembrane protein with a C-terminus extracellular TNF homology domain (THD) that assembles in homotrimers and regulates the course of the immune responses by signaling through 2 receptors, the herpes virus entry mediator (HVEM, TNFSFR14) and the lymphotoxin β receptor (LTβR, TNFSFR3). LIGHT is a membrane-bound protein transiently expressed on activated T cells, natural killer (NK) cells and immature dendritic cells that can be proteolytically cleaved by a metalloprotease and released to the extracellular milieu. The immunotherapeutic potential of LIGHT blockade was evaluated in vivo. Administration of an antagonist of LIGHT interaction with its receptors attenuated the course of graft-versus-host reaction and recapitulated the reduced cytotoxic activity of LIGHT-deficient T cells adoptively transferred into non-irradiated semiallogeneic recipients. The lack of LIGHT expression on donor T cells or blockade of LIGHT interaction with its receptors slowed down the rate of T cell proliferation and decreased the frequency of precursor alloreactive T cells, retarding T cell differentiation toward effector T cells. The blockade of LIGHT/LTβR/HVEM pathway was associated with delayed downregulation of interleukin-7Rα and delayed upregulation of inducible costimulatory molecule expression on donor alloreactive CD8 T cells that are typical features of impaired T cell differentiation. These results expose the relevance of LIGHT/LTβR/HVEM interaction for the potential therapeutic control of the allogeneic immune responses mediated by alloreactive CD8 T cells that can contribute to prolong allograft survival.