Macrophage ubiquitin-specific protease 2 modifies insulin sensitivity in obese mice

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.     

Malonylated flavonol glycosides from the petals of Clitoria ternatea

Three flavonol glycosides, kaempferol 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside, quercetin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside, and myricetin 3-O-(2",6"-di-O-alpha-rhamnosyl)-beta-glucoside were isolated from the petals of Clitoria ternatea cv. Double Blue, together with eleven known flavonol glycosides. Their structures were identified using UV, MS, and NMR spectroscopy. They were characterized as kaempferol and quercetin 3-(2(G)- rhamnosylrutinoside)s, kaempferol, quercetin, and myricetin 3-neohesperidosides, 3-rutinosides, and 3-glucosides in the same tissue. In addition, the presence of myricetin 3-O-(2"-O-alpha-rhamnosyl-6"-O-malonyl)-beta-glucoside was inferred from LC/MS/MS data for crude petal extracts. The flavonol compounds identified in the petals of C. ternatea differed from those reported in previous studies.     

Role of genetic variations of <Emphasis Type="Italic">chitinase 3</Emphasis>-<Emphasis Type="Italic">like 1</Emphasis> in bronchial asthmatic patients

Background

Single nucleotide polymorphisms (SNPs) in chitinase 3-like 1 (CHI3L1) are associated with bronchial severity and pulmonary function. CHI3L1 proteins are involved in both innate and adaptive immune responses; however, to date, the correlation of these SNPs and their age of onset of bronchial asthma has not been demonstrated.

Methods

To address the role of these genetic variations, 390 patients with well-controlled bronchial asthma and living in Japan were recruited, genotyped, and had a pulmonary function test performed on them in this study. To analyze the concentration levels of CHI3L1 protein, bronchial lavage fluids were examined.

Results

Forced expiratory volume in one second, %predicted (%FEV1), was significantly decreased in homozygotes of rs1214194 compared to heterozygotes and wild type. The age of onset of adult bronchial asthma was significantly younger in GG homozygotes of rs4950928 and AA homozygotes of rs1214194 than in the other two genotypes. The concentration of CHI3L1 protein in bronchial lavage fluid increased in both homozygotes of rs4950928 and rs1214194.

Conclusions

Our study demonstrated that the homozygotes of rs4950928 and rs1214194 of CHI3L1 might predict an early onset of bronchial asthma and have the propensity to promote airway remodeling.Trial registration JMA-IIA00045 remodeling-ICS

     

CYP306A1, a cytochrome P450 enzyme, is essential for ecdysteroid biosynthesis in the prothoracic glands of Bombyx and Drosophila

Ecdysteroids mediate a wide variety of developmental and physiological events in insects. In the postembryonic development of insects, ecdysone is synthesized in the prothoracic gland (PG). Although many studies have revealed the biochemical and physiological properties of the enzymes for ecdysteroid biosynthesis, most of the molecular identities of these enzymes have not been elucidated. Here we describe an uncharacterized cytochrome P450 gene, designated Cyp306a1, that is essential for ecdysteroid biosynthesis in the PGs of the silkworm Bombyx mori and fruit fly Drosophila melanogaster. Using the microarray technique for analyzing gene expression profiles in PG cells during Bombyx development, we identified two PG-specific P450 genes whose temporal expression patterns are correlated with changes in ecdysteroid titer during development. Amino acid sequence analysis showed that one of the Bombyx P450 genes belongs to the CYP306A1 subfamily. The temporal and spatial expression pattern of the Drosophila Cyp306a1 homolog is essentially the same as that of Bombyx Cyp306a1. We also found that Drosophila Cyp306a1 is disrupted in the phantom (phm) mutant, known also as the Halloween mutant. The morphological defects and decreased expression of ecdysone-inducible genes in phm suggest that this mutant cannot produce a high titer of ecdysone. Finally we demonstrate that S2 cells transfected with Cyp306a1 convert ketodiol to ketotriol via carbon 25 hydroxylation. These results strongly suggest that CYP306A1 functions as a carbon 25 hydroxylase and has an essential role in ecdysteroid biosynthesis during insect development.     

Functional heterogeneity of bone morphogenetic protein receptor-II mutants found in patients with primary pulmonary hypertension

Germline mutations in the BMPR2 gene encoding bone morphogenetic protein (BMP) type II receptor (BMPR-II) have been reported in patients with primary pulmonary hypertension (PPH), but the contribution of various types of mutations found in PPH to the pathogenesis of clinical phenotypes has not been elucidated. To determine the biological activities of these mutants, we performed functional assays testing their abilities to transduce BMP signals. We found that the reported missense mutations within the extracellular and kinase domains of BMPR-II abrogated their signal-transducing abilities. BMPR-II proteins containing mutations at the conserved cysteine residues in the extracellular and kinase domains were detected in the cytoplasm, suggesting that the loss of signaling ability of certain BMPR-II mutants is due at least in part to their altered subcellular localization. In contrast, BMPR-II mutants with truncation of the cytoplasmic tail retained the ability to transduce BMP signals. The differences in biological activities among the BMPR-II mutants observed thus suggest that additional genetic and/or environmental factors may play critical roles in the pathogenesis of PPH.     

Crystal Structure of Family 14 Polysaccharide Lyase with pH-dependent Modes of Action

The Chlorella virus enzyme vAL-1 (38 kDa), a member of polysaccharide lyase family 14, degrades the Chlorella cell wall by cleaving the glycoside bond of the glucuronate residue (GlcA) through a β-elimination reaction. The enzyme consists of an N-terminal cell wall-attaching domain (11 kDa) and a C-terminal catalytic module (27 kDa). Here, we show the enzyme characteristics of vAL-1, especially its pH-dependent modes of action, and determine the structure of the catalytic module. vAL-1 also exhibited alginate lyase activity at alkaline pH, and truncation of the N-terminal domain increased the lyase activity by 50-fold at pH 7.0. The truncated form vAL-1(S) released di- to hexasaccharides from alginate at pH 7.0, whereas disaccharides were preferentially generated at pH 10.0. This indicates that vAL-1(S) shows two pH-dependent modes of action: endo- and exotypes. The x-ray crystal structure of vAL-1(S) at 1.2 Å resolution showed two antiparallel β-sheets with a deep cleft showing a β-jelly roll fold. The structure of GlcA-bound vAL-1(S) at pH 7.0 and 10.0 was determined: GlcA was found to be bound outside and inside the cleft at pH 7.0 and 10.0, respectively. This suggests that the electric charges at the active site greatly influence the binding mode of substrates and regulate endo/exo activity. Site-directed mutagenesis demonstrated that vAL-1(S) has a specific amino acid arrangement distinct from other alginate lyases crucial for catalysis. This is, to our knowledge, the first study in which the structure of a family 14 polysaccharide lyase with two different modes of action has been determined.