Secretory regulation of 19-hydroxyandrostenedione in normal man.

To evaluate the secretory regulation of 19-hydroxyandrostenedione (19-OH-AD), its plasma concentration was measured before and after stimulation and inhibition tests for the ACTH-adrenal axis and the renin-angiotensin system in 50 normal subjects. Basal levels of plasma 19-OH-AD did not correlate with either those of plasma renin activity (PRA) or the plasma aldosterone concentration (PAC), but positively correlated with those of plasma cortisol. Plasma 19-OH-AD was stimulated by 0.25 mg ACTH-(1-24) and was suppressed by 1 mg dexamethasone (DEX) as were plasma cortisol and PAC. On the other hand, with 2-h standing alone or iv 40 mg furosemide plus 2-h standing, plasma 19-OH-AD and cortisol did not increase but PRA and PAC did. With iv furosemide plus 2-h standing with 3 mg DEX pretreatment, plasma 19-OH-AD and cortisol did not respond either, but PRA and PAC increased. With 25 mg oral captopril following 1-h standing with 3 mg DEX pretreatment, plasma 19-OH-AD and cortisol did not change but PAC decreased. These results indicate that the secretion of 19-OH-AD is mainly under the control of the ACTH-adrenal axis rather than the renin-angiotensin system.     

Splicing factor 3b subunit 4 binds BMPR-IA and inhibits osteochondral cell differentiation

Bone morphogenetic protein (BMP)-2/4 play critical roles in early embryogenesis and skeletal development. BMP-2/4 signals conduct into cells via two types of serine/threonine kinase receptors, known as BMPR-I (IA and IB) and BMPR-II. Here we identified splicing factor 3b subunit 4 (SF3b4) as a molecule that interacts with BMPR-IA, using a yeast two-hybrid screening with a human fetal brain cDNA library. Co-immunoprecipitation/immunoblot analysis confirmed their interaction in mammalian cells. By separation of the cell components, SF3b4 was present in the cell membrane fraction with BMPR-IA as well as in the nucleus. Overexpression of SF3b4 inhibited BMP-2-mediated osteogenic and chondrocytic differentiation of C2C12 and ATDC5 cells, respectively, and the endogenous expression level of SF3b4 decreased during differentiation in ATDC5 cells. By reporter gene assay, SF3b4 suppressed Id reporter gene activity, specific to the Smad1/5/8 pathway, but not TGFbeta-mediated reporter gene activity. Biotin labeling of the cell surface proteins followed by their immunoblot revealed that SF3b4 decreased the cell surface BMPRI-A levels. Further analysis by molecular modeling of the intracellular domain of BMPR-IA, coupled with binding studies of its several mutants, indicated that the site(s) for SF3b4 binding is not directly associated with the C-terminal lobe and the activation segment. Taken together, these results suggest that SF3b4, known to be localized in the nucleus and involved in RNA splicing, binds BMPR-IA and specifically inhibits BMP-mediated osteochondral cell differentiation.     

Identification of putative metabolites of docosahexaenoic acid as potent PPARgamma agonists and antidiabetic agents

We found that putative metabolites of docosahexaenoic acid (DHA) are strong PPARgamma activators and potential antidiabetic agents. We designed DHA derivatives based on the crystal structure of PPARgamma, synthesized them and evaluated their activities in vitro and in vivo. The efficacy of 5E-4-hydroxy-DHA 2a as a PPARgamma activator was about fourfold stronger than that of pioglitazone. Furthermore, the 4-keto derivative (10b) showed antidiabetic activity in animal models without producing undesirable effects such as obesity and hepatotoxicity.     

Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (Amadori-PE; deoxy-D-fructosyl phosphatidylethanolamine), because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo is still unclear. Consequently, we developed an analysis method of Amadori-PE using a quadrupole/linear ion-trap mass spectrometer, the Applied Biosystems QTRAP. In positive ion mode, collision-induced dissociation of Amadori-PE produced a well-characterized diglyceride ion ([M+H-303]+) permitting neutral loss scanning and multiple reaction monitoring (MRM). When lipid extract from diabetic plasma was infused directly into the QTRAP, Amadori-PE molecular species could be screened out by neutral loss scanning. Interfacing liquid chromatography with QTRAP mass spectrometry enabled the separation and determination of predominant plasma Amadori-PE species with sensitivity of approximately 0.1 pmol/injection in MRM. The plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15-0.29 mol% in diabetic patients. Furthermore, plasma Amadori-PE levels were positively correlated with PCOOH (a maker for oxidative stress). These results show the involvement between lipid glycation and lipid peroxidation in diabetes pathogenesis.     

Role of the kinesin-2 family protein, KIF3, during mitosis

During mitosis, kinesin and dynein motor proteins play critical roles in the equal segregation of chromosomes between two daughter cells. Kinesin-2 is composed of two microtubule-based motor subunits, KIF3A/3B, and a kinesin-associated protein known as KAP3, which links KIF3A/3B to cargo that is carried to cellular organelles along microtubules in interphase cells. We have shown here that the kinesin-2 complex is localized with components of the mitotic apparatus such as spindle microtubules and centrosomes. Furthermore, we found that expression of a mutant KIF3B, which is able to associate with KIF3A but not KAP3 in NIH3T3 cells, caused chromosomal aneuploidy and abnormal spindle formation. Our data suggested that the kinesin-2 complex plays an important role not only in interphase but also in mitosis.     

The endosymbiont Wolbachia increases insulin/IGF-like signalling in Drosophila

Insulin/IGF-like signalling (IIS) is an evolutionarily conserved pathway that has diverse functions in multi-cellular organisms. Mutations that reduce IIS can have pleiotropic effects on growth, development, metabolic homeostasis, fecundity, stress resistance and lifespan. IIS is also modified by extrinsic factors. For instance, in the fruitfly Drosophila melanogaster, both nutrition and stress can alter the activity of the pathway. Here, we test experimentally the hypothesis that a widespread endosymbiont of arthropods, Wolbachia pipientis, can alter the degree to which mutations in genes encoding IIS components affect IIS and its resultant phenotypes. Wolbachia infection, which is widespread in D. melanogaster in nature and has been estimated to infect 30 per cent of strains in the Bloomington stock centre, can affect broad aspects of insect physiology, particularly traits associated with reproduction. We measured a range of IIS-related phenotypes in flies ubiquitously mutant for IIS in the presence and absence of Wolbachia. We show that removal of Wolbachia further reduces IIS and hence enhances the mutant phenotypes, suggesting that Wolbachia normally acts to increase insulin signalling. This effect of Wolbachia infection on IIS could have an evolutionary explanation, and has some implications for studies of IIS in Drosophila and other organisms that harbour endosymbionts.     

Detection of cytomegalovirus in urine of HIV-infected patients by DNA-DNA hybridization comparison with virus isolation, immunofluorescence and immunoperoxidase.

Immunofluorescence and immunoperoxidase test directed against early viral antigens, and DNA-DNA hybridization were compared with viral isolation for their abilities to detect Cytomegalovirus (CMV) in the urine of 89 HIV infected patients. From the 100 urine samples collected, 70 were found positive by at least one method. Considering viral isolation as the "gold standard" technique, immunofluorescence and immunoperoxidase had a sensitivity of 92.3% and 88% respectively, with a specificity in both cases of 95%. DNA-DNA hybridization showed a sensitivity of 90% but with lower (60%) specificity. All of the three assays were effective in detecting CMV from urine and the technical advantage of each is discussed.     

19-hydroxyandrostenedione does not modulate [3H]aldosterone binding to human mononuclear leucocytes and rat renal cytosol

To verify the aldosterone amplifying action of 19-hydroxyandrostenedione (19-OH-AD), we investigated [3H]aldosterone and [3H]19-OH-AD binding to type I (mineralocorticoid) receptor in the renal cytosol of adrenalectomized and ovariectomized rat, and human mononuclear leucocytes (MNL). In the [3H]aldosterone binding study, the cytosol was incubated with [3H]aldosterone and 200-fold RU28362 (11 beta,17 beta-dihydroxy-6-methyl,17 alpha-(1-propynyl)-androsta-1,4,6- trien-3-one), a pure glucocorticoid, with or without 19-OH-AD. Scatchard plots of [3H]aldosterone binding to cytosol with 0.2 or 20 nM 19-OH-AD or without 19-OH-AD were linear. Dissociation constants (Kd) and maximum bindings (Bmax) without 19-OH-AD, and with 0.2 and 20 nM 19-OH-AD were: 0.71 +/- 0.03 nM and 23.0 +/- 3.4 fmol/mg protein (mean +/- SD, n = 3), 0.72 +/- 0.05 nM and 23.1 +/- 2.3 fmol/mg protein (n = 3), and 0.77 +/- 0.04 nM and 22.9 +/- 4.8 fmol/mg protein (n = 3), respectively. 19-OH-AD did not significantly change the Kd and Bmax of [3H]aldosterone binding. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM [3H]aldosterone bound to cytosol. In human MNL, Scatchard plots of [3H]aldosterone binding with both 0.2 and 20 nM 19-OH-AD and without 19-OH-AD were linear. Kd and Bmax were, respectively, 1.00 nM and 780 sites/cell in the absence of 19-OH-AD, and 1.07 nM and 774 sites/cell in the presence of 0.2 nM 19-OH-AD. Without 19-OH-AD they were, respectively, 0.95 nM and 551 sites/cell, and 1.10 nM and 560 sites/cell with 20 nM 19-OH-AD. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM of [3H]aldosterone bound to MNL. In both tissues, there was no obvious specific binding of [3H]19-OH-AD within the range of 1-60 nM. The above results suggest that the amplifying effect of 19-OH-AD on aldosterone mineralocorticoid action may not occur at the binding site of aldosterone to type I receptor, and that 19-OH-AD itself may not have any direct or indirect mineralocorticoid actions on the steroid receptor-mediated process in the rat kidney and human MNL.     

A large-scale genetic association study of ossification of the posterior longitudinal ligament of the spine

Research to date has identified several genes that are implicated in the etiology of ossification of the posterior longitudinal ligament of the spine (OPLL); however, their pathogenetic relevance remains obscure. The aim of this study is to identify susceptibility genes for OPLL through a large-scale case–control association study and to re-examine previously reported associations. A total of 109 single nucleotide polymorphisms (SNPs) in 35 candidate genes were genotyped for 711 sporadic OPLL patients and 896 controls. The differences in allelic and genotypic distribution between patients and controls were assessed using the χ ² test with Bonferroni’s correction. We also analyzed the association by separating patients into subgroups according to sex, age and the number of ossified vertebrae. The nominal P values fell below 0.05 for five SNPs in three genes. An intronic SNP in the TGF3 gene (P=0.00040) showed the most significant association. Previously reported associations of COL11A2, NPPS and TGFB1 with OPLL could not be reproduced. Further, no significant associations were detected in stratified analyses based on sex, age or the number of ossified vertebrae. TGFB3 warrants further investigation because it is located within a genomic region that has been positively linked with OPLL.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Taizo Horikoshi and Koichi Maeda contributed equally to this work.