Retardation of removal of radiation-induced apoptotic cells in developing neural tubes in macrophage galactose-type C-type lectin-1-deficient mouse embryos

MGL1/CD301a is a C-type lectin that recognizes galactose and N-acetylgalactosamine as monosaccharides and is expressed on limited populations of macrophages and dendritic cells at least in adult mice. In this study, pregnant mice with Mgl1+/- genotype were mated with Mgl1+/- or Mgl1-/- genotype males, and the embryos were used to assess a hypothesis that this molecule plays an important role in the clearance of apoptotic cells. After X-ray irradiation at 1 Gy of developing embryos at 10.5 days post coitus (d.p.c.), the number of Mgl1-/- pups was significantly reduced as compared with Mgl1+/+ pups. Distributions of MGL1-positive cells, MGL2-positive cells, and apoptotic cells were histologically examined in irradiated Mgl1+/+ embryos. MGL1-positive cells were detected in the neural tube in which many cells undergo apoptosis, whereas MGL2-positive cells were not observed. Biotinylated recombinant MGL1 bound a significant portion of the apoptotic cells. When Mgl1+/+ and Mgl1-/- embryos were examined for the presence of apoptotic cells, similar numbers of apoptotic cells gave rise, but the clearance of these cells was slower in Mgl1-/- embryos than in Mgl1+/+ embryos. These results strongly suggest that MGL1/CD301a is involved in the clearance of apoptotic cells. This process should be essential in the repair and normal development of X-ray-irradiated embryos.     

The Calvin cycle in cyanobacteria is regulated by CP12 via the NAD(H)/NADP(H) ratio under light/dark conditions

In Synechococcus PCC7942 cells grown in the dark, the concentrations of NAD(H) and NADP(H) were 128+/-2.5 and 483+/-4.0 microm, respectively, while those in the cells under light conditions were 100+/-5.0 and 649+/-7.0 microm, respectively. Analysis of gel filtration indicated that the change of the ratio of NADP(H) to NAD(H) in cyanobacterial cells under light/dark conditions controls the reversible dissociation of the PRK/CP12/GAPDH complex (approximately 520 kDa) consisting of phosphoribulokinase (PRK), CP12, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). S. 7942 CP12 lacked the two Cys residues essential for formation of the N-terminal peptide loop in the CP12 of higher plants, but the N-terminal region of S. 7942 CP12 had the ability to be associated with PRK. The growth of mutant cells in which the CP12 gene was disrupted by a kanamycin resistance cartridge gene was almost the same as that of wild-type cells under continuous light conditions. However, under the light/dark cycle (12 h/12 h), the growth of CP12-disrupted mutant cells was significantly inhibited compared with that of wild-type cells. The mutant cells showed a decreased rate of O2 consumption and an increased level of ribulose 1,5-bisphosphate compared with wild-type cells in the dark. These data suggest that under light and dark conditions, the oligomerization of CP12 with PRK and GAPDH regulates the activities of both enzymes and thus the carbon flow from the Calvin cycle to the oxidative pentose phosphate cycle.     

Novel and orally bioavailable inducible nitric oxide synthase inhibitors: synthesis and evaluation of optically active 4,5-dialkyl-2-iminoselenazolidine derivatives

We have previously reported that (4R,5R)-5-ethyl-2-imino-4-methylthiazolidine (3) strongly inhibits inducible nitric oxide synthase (iNOS). In a successive search for strong and selective iNOS inhibitors, we, herein, describe the synthesis of the selenium analogue of 3 (4: ES-2133) and its related optically active compounds and examine their in vitro and in vivo inhibitory activity against iNOS. In addition, an alternative synthetic method to the selected compound 4 and its pharmacokinetic profile is also reported.     

Tubulin-polymerization inhibitors derived from thalidomide

2-(2,6-Diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33), which was obtained during our previous structural development studies on thalidomide, was revealed to possess potent tubulin-polymerization-inhibiting activity, comparable to that of the known tubulin-polymerization inhibitors, rhizoxin and colchicine. A major metabolite of thalidomide, 5-hydroxythalidomide, which possesses a hydroxyl group at the position corresponding to that of 5HPP-33, also showed moderate inhibitory activity.     

Syntheses and properties of the major hydroxy metabolites in humans of blonanserin AD-5423, a novel antipsychotic agent

Two major metabolites in humans of blonanserin, 2-(4-ethyl-1-piperazinyl)-4-(4-fluorophenyl)-5,6,7,8,9,10-hexahydrocycloocta-[b]pyridine (code name AD-5423), were synthesized. The first, 7-hydroxylated AD-5423, was synthesized through a four-step process starting from 4-fluorobenzoylacetonitrile (1), and the second, 8-hydroxylated AD-5423, a nine-step process also from 1. The optical resolution, structures, and receptor binding properties of the metabolites were documented.     

Nuclear transfer of perchloric acid-soluble protein by endoplasmic reticulum stressors

Perchloric acid-soluble protein (PSP) is highly conserved during evolution from bacteria to mammals. Although PSP has been recognized as an inhibitor of translation and proliferation in vitro, its precise biological role has not yet been elucidated. Since we previously found similar distributions for PSP and the endoplasmic reticulum (ER) and Golgi complex, the intracellular distribution of PSP was analyzed in more detail. Immunofluorescence studies indicated that PSP co-localized with the ER and Golgi complex, since the distribution pattern of PSP was well matched to both of these organelles. An immunoelectron microscopic study revealed PSP was located not only in the cytosol but also on the surface of the outer ER membrane. Since PSP was present on the ER, we speculated that it may be associated with ER function. Therefore, we analyzed whether or not the ER stress response, which is one of the ER functions, affected PSP expression. The results showed that various ER stressors (thapsigargin, A23187, tunicamycin, brefeldin A, and cisplatin) provoked a dramatic change in the localization of PSP from outside of the nucleus to inside the nucleus within 3 h. Moreover, the ER stressors induced PSP expression. These results suggest that PSP is involved in the cellular response to ER stressors, and that the change in localization of PSP from the ER to the nucleus may be associated with ER stress responses.     

An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool

The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin alpha-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3' end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21-25 kDa) toxic to host cells or two antibody fragments (25-36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities.     

Comparative study of eye defective worm 'menashi' and regenerating wild-type in planarian, Dugesia ryukyuensis

Inbreeding of the sexualized planarian, Dugesia ryukyuensis, produces eye-defective worms, menashi, in the F1 population. To study the effects of this mutation on the eye, we observed the eye-region of menashi using electron microscopy and compared it with the regenerating eye in wild-type worms. The intact eye of wild-type planarians consisted of a few pigment cells and a number of visual cells. Pigment cells containing spherically-shaped electron-dense melanosomes contacted each other and enclosed rhabdomes of visual cells. Rhabdomes had numerous tubular microvilli extending radially and touching the pigment cells. However, in menashi, various lengths of tubular microvilli were irregularly distributed near the pigment cells, which contained numerous electron-lucent premelanosomes, and no adhesive structures were found between the pigment cells. The premelanosomes of menashi were equal in size to those seen after 2 days of regeneration in wild-type planarians and were similar in maturation to those found after 3 days of regeneration in wild-type planarian. These results suggest that menashi is defective in the mechanism(s) of developing pigment granules and constructing visual cells. These findings also suggest that pigment cells in menashi are defective in the mechanism(s) involved with cell adhesion.