Cloning of rat ABCA7 and its preferential expression in platelets

We cloned the full-length cDNA of a rat orthologue of ABCA7 (rABCA7) from rat platelets. The cDNA of rABCA7 is 6510bp in length and encodes a protein of 2170 amino acids. The amino acid sequence of rABCA7 exhibits homology to those of mouse ABCA7 (92.5% identical in amino acid sequence) and human ABCA7 (76.6%). We obtained two clones of monoclonal antibodies against rABCA7 recognizing different epitopes. Analysis of CHO cells stably expressing rABCA7 by confocal laser-scanning microscopy indicated that rABCA7 is mainly located in the plasma membrane. Western blot analysis of rat tissues revealed that rABCA7 was preferentially expressed in platelets and that its apparent molecular mass was 250kDa. This is the first report of the tissue distribution of rABCA7 at the protein level and is the first reported case of ABC transporters being expressed in platelets, suggesting their important role in platelet function.     

Bone morphogenetic protein signaling in articular chondrocyte differentiation

Articular chondrocytes progressively undergo dedifferentiation into a spindle-shaped mesenchymal cellular phenotype in monolayers. Chondrocyte dedifferentiation is stimulated by retinoic acid. On the other hand, bone morphogenic proteins (BMPs) stimulate differentiation of chondrocytes. We examined the mechanism of effects of BMP in chondrocyte differentiation with use of a recombinant adenovirus vector system. Constitutively active forms of BMP type I receptors (BMPR-IA and BMPR-IB) and those of activin receptor-like kinase (ALK)-1 and ALK-2 maintained differentiation of chondrocytes in the presence of retinoic acid. The BMP receptor-regulated signaling substrates, Smad1/5, weakly induced chondrocyte differentiation; the effects of Smad1/5 were enhanced by BMP-7 treatment. Inhibitory Smad, Smad6, blocked increase of expression of chondrocyte markers by BMP-7 in a dose-dependent manner. SB202190, a p38 mitogen-activated protein kinase inhibitor, inhibited this effect of BMP-7; however, since SB202190 suppressed phosphorylation of Smad1/5, this may be due to blockade of BMP receptor activation. These results together strongly suggest that induction of chondrocyte differentiation by BMP-7 is regulated by Smad pathways.     

Mouse uterine epithelial apoptosis is associated with expression of mitochondrial voltage-dependent anion channels,release of cytochrome C from mitochondria,and the ratio of Bax to Bcl-2 or Bcl-X

The release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members and is considered to take place through voltage-dependent anion channels (VDACs) on the outer membranes of mitochondria, results in activation of effector caspases, such as caspase-3, which induce apoptosis. We studied the involvement of the mitochondrial apoptosis pathway in uterine epithelial apoptosis. Estradiol-17beta pellets were implanted into ovariectomized mice and removed 4 days later (Day 0). The apoptotic index (percentage of apoptotic cells) of the luminal epithelium increased markedly, peaking on Day 2, whereas that of the glandular epithelium increased much less. Expression of VDAC1, 2, and 3 mRNAs increased in the luminal epithelium in correlation with the apoptotic index of the luminal epithelium. No increases in VDAC1, 2, and 3 mRNA levels were observed in the stroma or muscle, where no apoptosis occurs. VDAC1 protein levels in the uterus also correlated well with the apoptotic index of the luminal epithelium. In addition, the apoptotic index showed good correlation with the release of cytochrome c from mitochondria, activation of caspase-3, which was immunohistochemically detected only in the epithelium, and the mRNA and protein ratios of Bax:Bcl-2 and Bax:Bcl-X in the uterus. The present results suggest that the release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members, plays a role in uterine epithelial apoptosis after estrogen deprivation. The increase in VDAC expression may facilitate the release of cytochrome c during apoptosis.     

In vitro induction and transplantation of eye during early Xenopus development

A vertebrate eye was induced via a series of coordinated inductive interactions. Here, we describe a novel in vitro system to induce eye formation at high frequency using Xenopus early gastrulae. The eye formed in vitro is morphologically similar to the normal eye. When the in vitro eye was transplanted into a stage-33 tadpole, the optic nerve was seen extending from the grafted eye to the tectum of the host brain and the in vitro eye graft was retained after metamorphosis. In addition, we transplanted the eye formed in vitro into a tadpole with both eyes removed. The resultant juvenile frogs could perceive brightness using the grafted eye and thereby control their skin color, suggesting that the eye formed in vitro could function normally.     

Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway

FcR provides a critical link between ligands and effector cells in immune complex diseases. Emerging evidence reveals that angiotensin (Ang)II exerts a wide variety of cellular effects and contributes to the pathogenesis of inflammatory diseases. In anti-glomerular basement membrane Ab-induced glomerulonephritis (GN), we have previously noted that FcR-deficient mice (gamma(-/-)) surviving from lethal initial damage still developed mesangial proliferative GN, which was drastically prevented by an AngII type 1 receptor (AT1) blocker. We further examined the mechanisms by which renin-Ang system (RAS) participates in this immune disease. Using bone marrow chimeras between gamma(-/-) and AT1(-/-) mice, we found that glomerular injury in gamma(-/-) mice was associated with CD4(+) T cell infiltration depending on renal AT1-stimulation. Based on findings in cutaneous delayed-type hypersensitivity, we showed that AngII-activated renal resident cells are responsible for the recruitment of effector T cells. We next examined the chemotactic activity of AngII-stimulated mesangial cells, as potential mechanisms coupling RAS and cellular immunity. Chemotactic activity for T cells and Th1-associated chemokine (IFN-gamma-inducible protein-10 and macrophage-inflammatory protein 1alpha) expression was markedly reduced in mesangial cells from AT1(-/-) mice. Moreover, this activity was mainly through calcineurin-dependent NF-AT. Although IFN-gamma-inducible protein-10 was NF-kappaB-dependent, macrophage-inflammatory protein 1alpha was dominantly regulated by NF-AT. Furthermore, AT1-dependent NF-AT activation was observed in injured glomeruli by Southwestern histochemistry. In conclusion, our data indicate that local RAS activation, partly via the local NF-AT pathway, enhances the susceptibility to T cell-mediated injury in anti-glomerular basement membrane Ab-induced GN. This novel mechanism affords a rationale for the use of drugs interfering with RAS in immune renal diseases.     

Synthesis of milbemycins beta9 and beta10 from milbemycins A3 and A4 and their biological activities

Chemical derivation methods were used to prepare milbemycins beta9 and beta10 from milbemycins A3 and A4. Their acaricidal activities were also assessed against the organophosphorus-sensitive two-spotted spider mite (Tetranychus urticae) on primary leaves of cowpea plants (Vigna sinesis Savi species) by spraying.