Synthesis and optimization of 4,5,6,7-tetrahydrooxazolo[4,5-c]pyridines as potent and orally-active metabotropic glutamate receptor 5 negative allosteric modulators

We describe here the design, synthesis and characterization of a series of 4,5,6,7-tetrahydrooxazolo[4,5-c]pyridines as metabotropic glutamate receptor (mGluR) 5 negative allosteric modulators (NAMs). Optimization of the substituents led to the identification of several compounds with good pharmacokinetic profiles, including long half life and high oral bioavailability, in both rats and monkeys. The receptor occupancy test in the rat cortex revealed favorable brain penetration of these compounds. The reprsentative compound 13 produced oral antidepressant-like effect in the rat forced swimming test (MED: 0.3 mg/kg, q.d.).     

Neuromyelitis optica: Passive transfer to rats by human immunoglobulin

Recurrent attacks of optic neuritis and myelitis are the hallmarks of both neuromyelitis optica (NMO) and multiple sclerosis (MS). NMO immunoglobulin G (NMO-IgG), which recognizes astrocytic aquaporin-4 (AQP4) water channels, is a specific serum autoantibody that distinguishes NMO from MS. The pathogenic role of the anti-AQP4 antibody (AQP4-Ab, NMO-IgG) in NMO has been speculated based on several studies in vitro. The aim of this study was to demonstrate the pathogenicity of AQP4-Ab in vivo. We obtained IgG from patients who underwent therapeutic plasmapheresis, and developed an animal model by passive transfer of IgG to rats. The active lesions of the rats exhibited pathological characteristics strikingly similar to those of NMO, marked by astrocytic loss and perivascular deposition of immunoglobulin and complements. These findings provide the first evidence of the pathogenicity of AQP4-Ab in vivo and support the therapeutic efficacy of eliminating the antibodies by plasmapheresis.     

Use of colloidal gold and ruthenium red in stereo high-voltage electron microscopic study of Con A-binding sites in mouse macrophages

Concanavalin A (Con A)-binding sites were labeled with colloidal gold (CG), stained with ruthenium red, and observed under a high-voltage electron microscope. Mouse peritoneal macrophages were labeled by the indirect Con A/CG labeling method at 0 degree C. After washing, some of the cells were incubated in phosphate-buffered saline (PBS) at 37 degrees C. The specimens were then stained with ruthenium red, to enhance the contrast of the cell surface, and embedded in Epon. Sections (0.3 approximately 3 micron thick) were cut and examined by high-voltage electron microscopy at accelerating voltages of 200 approximately 1,000 kV. Staining with ruthenium red provided a strong contrast of the cell surface and the invaginating tubules beneath it against the cytoplasm; in thick sections, both of them were clearly seen by stereomicroscopy. CG particles which represented Con A-binding sites were also sufficiently electron dense to be recognized by high-voltage electron microscopy of thick sections. The two- and three-dimensional distribution of CG particles on the ruthenium-red-positive cell surface was clearly visualized. At 0 degree C, Con A-binding sites were randomly distributed on the cell surface. The redistribution and endocytosis of Con A-binding sites were seen at 37 degrees C. The three-dimensional organization of membrane invagination, which represented the process of endocytosis, was clearly seen by stereomicroscopy. The combination of CG labeling and ruthenium red staining is a useful method for high-voltage electron microscopic analysis of the two- and three-dimensional distribution of CG-labeled ligands on the cell surface in thick sections.     

Involvement of actin filaments in mouse testicular cord organization in vivo and in vitro.

Involvement of actin filaments in mouse fetal testicular differentiation was examined in vivo and in vitro. During testicular cord formation in vivo, actin filaments accumulated in the basal cytoplasm of Sertoli cells. Addition of cytochalasin D (CD) to organ cultures of undifferentiated gonadal primordia significantly inhibited testicular cord formation. In brief, treatment with 25 ng/ml CD induced the formation of slender testicular cords, and treatment with 50 ng/ml largely inhibited cord formation in the explants. However, development and growth of the testicular parenchyma and Leydig cell differentiation occurred in the presence of CD. By electron microscopic and immunohistochemical examinations, it became clear that CD also affected formation of the basal lamina and accumulation of vimentin filaments in Sertoli cells. On the other hand, treatment with colcemid at 12.5 or 15 ng/ml prevented growth of the testicular parenchyma and development of interstitial regions. Interestingly, testicular cords formed under this condition. These results indicate that the basal actin filaments of Sertoli cells may play an important role in testicular cord formation, especially Sertoli cell polarization. Cell mitosis and/or microtubules, on the other hand, may not be directly involved in this process.     

Complete nucleotide sequence of the <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>D. Don. chloroplast genome and comparative chloroplast genomics: diversified genomic structure of coniferous species

Background  

The recent determination of complete chloroplast (cp) genomic sequences of various plant species has enabled numerous comparative analyses as well as advances in plant and genome evolutionary studies. In angiosperms, the complete cp genome sequences of about 70 species have been determined, whereas those of only three gymnosperm species, Cycas taitungensis, Pinus thunbergii, and Pinus koraiensis have been established. The lack of information regarding the gene content and genomic structure of gymnosperm cp genomes may severely hamper further progress of plant and cp genome evolutionary studies. To address this need, we report here the complete nucleotide sequence of the cp genome of Cryptomeria japonica, the first in the Cupressaceae sensu lato of gymnosperms, and provide a comparative analysis of their gene content and genomic structure that illustrates the unique genomic features of gymnosperms.