Synthesis and structure-activity relationship of RXR antagonists based on the diazepinylbenzoic acid structure

Synthesis and structure-activity relationship of RXR antagonists employing a diazepinylbenzoic acid scaffold are described. Of those antagonists, sulfonamide derivatives (6v and 6w) reveal a high antagonistic activity and good pharmacokinetic properties.     

Transplantation of engineered bone tissue using a rotary three-dimensional culture system

Bone is a complex, highly structured, mechanically active, three-dimensional (3-D) tissue composed of cellular and matrix elements. We previously published a report on in situ collagen gelation using a rotary 3-D culture system (CG–RC system) for the construction of large tissue specimens. The objective of the current study was to evaluate the feasibility of bone tissue engineering using our CG–RC system. Osteoblasts from the calvaria of newborn Wistar rats were cultured in the CG–RC system for up to 3 wk. The engineered 3-D tissues were implanted into the backs of nude mice and calvarial round bone defects in Wistar rats. Cell metabolic activity, mineralization, and bone-related proteins were measured in vitro in the engineered 3-D tissues. Also, the in vivo histological features of the transplanted, engineered 3-D tissues were evaluated in the animal models. We found that metabolic activity increased in the engineered 3-D tissues during cultivation, and that sufficient mineralization occurred during the 3 wk in the CG–RC system in vitro. One mo posttransplantation, the transplants to nude mice remained mineralized and were well invaded by host vasculature. Of particular interest, 2 mo posttransplantation, the transplants into the calvarial bone defects of rats were replaced by new mature bone. Thus, this study shows that large 3-D osseous tissue could be produced in vitro and that the engineered 3-D tissue had in vivo osteoinductive potential when transplanted into ectopic locations and into bone defects. Therefore, this system should be a useful model for bone tissue engineering.     

Serum levels of sRAGE, the soluble form of receptor for advanced glycation end products, are associated with inflammatory markers in patients with type 2 diabetes

Advanced glycation end products (AGEs) and their receptor (RAGE) play an important role in accelerated atherosclerosis in diabetes. We have recently found that the soluble form of RAGE (sRAGE) levels are significantly higher in type 2 diabetic patients than in nondiabetic subjects and positively associated with the presence of coronary artery disease in diabetes. In this study, we examined whether serum levels of sRAGE correlated with inflammatory biomarkers in patients with type 2 diabetes. Eighty-six Japanese type 2 diabetic patients (36 men and 50 women, mean age 68.4+/-9.6 years) underwent a complete history and physical examination, determination of blood chemistries, sRAGE, monocyte chemotactic protein-1 (MCP-1), adiponectin, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6). Univariate regression analysis showed that serum levels of sRAGE positively correlated with alanine aminotransferase (ALT) (r=0.437, P=0.0001), MCP-1 (r=0.359, P=0.001), TNF-alpha (r=0.291, P=0.006), and hyperlipidemia medication (r=0.218, P=0.044). After multiple regression analyses, ALT (P<0.0001), MCP-1 (P=0.007), and TNF-alpha (P=0.023) remained significant. The present study demonstrates for the first time that serum levels of sRAGE are positively associated with MCP-1 and TNF-alpha levels in type 2 diabetic patients. These observations suggest the possibility that sRAGE level may become a novel biomarker of vascular inflammation in type 2 diabetic patients.     

Nucleotide substitutions in the acetolactate synthase genes of sulfonylurea-resistant biotypes of Monochoria vaginalis (Pontederiaceae)

Some point mutations in acetolactate synthase (ALS) confer resistance to ALS-inhibiting herbicides in weeds. To clarify the evolution of the herbicide resistance of Monochoria vaginalis, a weed in rice fields in Japan, the nucleotide sequences of four genes encoding ALS were surveyed in five sulfonylurea-resistant (SU-R) and five sulfonylurea-susceptible (SU-S) biotypes. In the ALS1 gene, two SU-R biotypes showed nucleotide substitutions changing Pro197 to Ser and Leu, respectively. In a different gene, ALS3, three other SU-R biotypes showed either of the two nonsynonymous nucleotide substitutions seen in ALS1. Only two biotypes geographically located distantly from each other shared the same mutation conferring SU resistance in the same gene. These patterns of nucleotide substitutions indicate that the SU-R phenotype was acquired independently by different biotypes. Nucleotide diversity values of the genes showing SU-R mutations were higher than those of ALS2 lacking any SU-R mutation and of a putative pseudogene, ALS4. This result suggests that the maintenance of nucleotide variability within target genes provides an opportunity for the evolution of SU-R phenotypes by herbicide-driven selection for mutations conferring resistance.     

Stimulation of the biosynthesis of the antibiotics lambertellols by the mycoparasitic fungus Lambertella corni-maris under the acidic conditions produced by its host fungus in vitro

The filamentous fungus, Lambertella corni-maris (L. corni-maris), a mycoparasite on Monilinia fructigena, produces the antibiotics, lambertellols A (1), B (2), and lambertellin (3), in a substantial amounts under acidic conditions, whereas these antibiotics were hardly detected when the fungus was cultured on a potato-sucrose (PS) medium without added acids. Our investigations also revealed that the host, M. fructigena, changed its surroundings into acidic conditions, suggesting that the acidic conditions acted as kairomones that stimulated the production of 1-3.     

Mineralization process during acellular cementogenesis in rat molars: a histochemical and immunohistochemical study using fresh-frozen sections

This study was designed to detect tissue non-specific alkaline phosphatase (TNSALP) by Azo-dye staining, calcium by glyoxal bis (2-hydroxyanil) (GBHA) staining, bone sialoprotein (BSP) and osteopontin (OPN) by immunoperoxidase staining in developing rat molars, and also to discuss the mineralization process during acellular cementogenesis. To restrain a reduction in histochemical and immunohistochemical reactions, fresh-frozen undemineralized sections were prepared. Where the epithelial sheath was intact, TNSALP reaction was observed in the dental follicle, but not in the epithelial sheath. With the onset of dentin mineralization, the BSP- and OPN-immunoreactive, initial cementum layer appeared. At this point, cementoblasts had shown intense TNSALP reaction and GBHA reactive particles (=calcium-GBHA complex) appeared on the root surface. With further development, the reaction of TNSALP and GBHA became weak on the root surface. Previous studies have shown that the initial cementum is fibril-poor and that matrix vesicles and calciferous spherules appear on the root surface only during the initial cementogenesis. The findings mentioned above suggest that: during the initial cementogenesis, cementoblasts release matrix vesicles which result in calciferous spherules, corresponding to the GBHA reactive particles. The calciferous spherules trigger the mineralization of the initial cementum. After principal fiber attachment, mineralization advances along collagen fibrils without matrix vesicles.     

Simultaneous quantification of seven prostanoids using liquid chromatography/tandem mass spectrometry: the effects of arachidonic acid on prostanoid production in mouse bone marrow-derived mast cells

We have developed a method for the simultaneous estimation of the levels of the prostanoids 6-keto prostaglandin (PG) Flalpha, PGB2, PGD2, PGE2, PGF2(alpha), PGJ2, and thromboxane (TX) B2 in blood- or serum-containing medium using liquid chromatography-tandem mass spectrometry. These prostanoids and their deuterium derivatives, which were used as internal standards, were subjected to solid-phase extraction using Empore C18 HD disk cartridges and analyzed in the selected reaction-monitoring mode. A linear response curve starting at 10 pg of prostanoid/tube was observed for each prostanoid. The accuracy of the method was demonstrated with samples containing known amounts of the prostanoids. Furthermore, we used this method to analyze the prostanoids produced in mouse bone marrow-derived mast cells stimulated with arachidonic acid, which resulted in the production of PGD2, PGE2, PGF2alpha, and TXB2. The results suggest that this simultaneous quantification method is useful for the analysis of the production of biomedically important prostanoids.