Mouse strain-dependent osteoclastogenesis in response to lipopolysaccharide

Bacterial lipopolysaccharide (LPS) is a potent stimulator of bone resorption in periodontitis. Co-culture systems of mouse calvaria-derived osteoblasts and bone marrow-derived preosteoclasts were used as an in vitro osteoclast differentiation. This study revealed that co-cultures using ddY or ICR mouse strain responded differently to LPS while responded equally to 1alpha,25(OH)2D3. Thus, the different response to LPS indicates dissimilarity of two mouse stains in their capacity for generating osteoclasts while the two mouse strains share the similarity in response to 1alpha,25(OH)2D3. To identify which cells between osteoblasts and preosteoclasts in the co-culture are responsible for the dissimilarity, the reciprocal co-cultures were performed between ddY and ICR mouse strains. The treatment of 1,25(OH)2D3 to ddY/ICR (osteoblasts from ddY/preosteoclasts from ICR) and ICR/ddY reciprocal co-cultures also showed the similarity. In case of LPS treatment, the results of ddY/ICR were similar to ddY/ddY and the results of the other reciprocal co-culture, ICR/ddY combination, were consistent with those of ICR/ICR. It suggests that the dissimilarity between the two mouse strains may resident in osteoblasts but not in preosteoclasts. Therefore, the osteoblast is responsible for mouse strain-dependent osteoclastogenesis in response to LPS. Although mouse models will continue to provide insights into molecular mechanisms of osteoclastogenesis, caution should be exercised when using different mouse strains, especially ddY and ICR strains as models for osteoclast differentiation.     

Identification of a functionally relevant signal peptide of mouse ficolin A

Mouse ficolin A is a plasma protein with lectin activity, and plays a role in host defense by binding carbohydrates, especially GlcNAc, on microorganisms. The ficolin A subunit consists of an N-terminal signal peptide, a collagen-like domain, and a C-terminal fibrinogen-like domain. In this study, we show that ficolin A can be synthesized and oligomerized in a cell and secreted into culture medium. We also identify a functionally relevant signal peptide of ficolin A by using MS/MS analysis to determine the N-terminal sequence of secreted ficolin A. When the signal peptide of mouse ficolin A was fused with enhanced green fluorescent protein (EGFP), EGFP was released into HEK 293 cell medium, suggesting that the signal peptide can efficiently direct ficolin A secretion. Moreover, our results suggest that the signal peptide of ficolin A has potential application for the production of useful secretory proteins.     

Comparative anatomy of the cheek muscles of Korean cobitid fishes (Ostariophysi: Cypriniformes), with the comments on their phylogenetic relationships

Cheek muscles of 16 species of all Korean cobitid species belong to six genera were examined and described in detail, with comments on their phylogenetic relationships made on the basis of characteristics of cheek muscles. Korean cobitids were divided into two groups, the Cobitis and Misgurnus groups, by five characters related to three cheek muscles (adductor mandibulae, preorbitalis, and retractor arcus palatine). The former group, comprising Cobitis, Iksookimia, Kichulchoia, Koreocobitis, and Niwaella, is defined by four apomorphies, and the latter, including Misgurnus, by a single apomorphy.     

Genetic diversity of <Emphasis Type="Italic">Salanx ariakensis</Emphasis> (Salangidae) from Korea and Japan inferred from AFLP

The genetic diversity and population structures within and between Korean and Japanese populations of Salanx ariakensis were investigated using AFLP (amplified fragment length polymorphism). Using seven primer pair sets, 411 fragments were amplified from 31 specimens of S. ariakensis, 393 fragments (96%) being polymorphic. The expected heterozygosities were 0.211 and 0.245 for the Japanese and Korean populations, respectively, levels similar to those of other freshwater and diadromous fishes, but lower than those of marine fishes. The difference in genetic diversity between the two populations may result from their different habitat sizes. Principal coordinate analysis using 393 polymorphic fragments resulted in specimens from each population being plotted separated, suggesting an absence (or little if present) gene flow between them since Tsushima Strait has been open. Therefore, each population of S. ariakensis is relevant from the conservation perspective and should at least be treated as a “management unit” for conservation purposes.     

Genetic variance modifies apoptosis susceptibility in mature oocytes via alterations in DNA repair capacity and mitochondrial ultrastructure

Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double-strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.     

Global analysis of exon creation versus loss and the role of alternative splicing in 17 vertebrate genomes

Association of alternative splicing (AS) with accelerated rates of exon evolution in some organisms has recently aroused widespread interest in its role in evolution of eukaryotic gene structure. Previous studies were limited to analysis of exon creation or lost events in mouse and/or human only. Our multigenome approach provides a way for (1) distinguishing creation and loss events on the large scale; (2) uncovering details of the evolutionary mechanisms involved; (3) estimating the corresponding rates over a wide range of evolutionary times and organisms; and (4) assessing the impact of AS on those evolutionary rates. We use previously unpublished independent analyses of alternative splicing in five species (human, mouse, dog, cow, and zebrafish) from the ASAP database combined with genomewide multiple alignment of 17 genomes to analyze exon creation and loss of both constitutively and alternatively spliced exons in mammals, fish, and birds. Our analysis provides a comprehensive database of exon creation and loss events over 360 million years of vertebrate evolution, including tens of thousands of alternative and constitutive exons. We find that exon inclusion level is inversely related to the rate of exon creation. In addition, we provide a detailed in-depth analysis of mechanisms of exon creation and loss, which suggests that a large fraction of nonrepetitive created exons are results of ab initio creation from purely intronic sequences. Our data indicate an important role for alternative splicing in creation of new exons and provide a useful novel database resource for future genome evolution research.     

TGFBIp/betaig-h3 protein: a versatile matrix molecule induced by TGF-beta

TGFBIp/βig-h3 protein is an extracellular matrix molecule initially cloned from human adenocarcinoma cells treated with TGF-β. Its precise function remains obscure but a number of studies have demonstrated it to be an intriguingly versatile molecule role in a wide range of physiological and pathological conditions. To date, the most extensively studied and reported action of TGFBIp/βig-h3 protein is in corneal dystrophy and several excellent reviews are available on this. Work from various laboratories on this molecule has compiled a tremendous amount of information over the past decade and a half. Here we review the current understanding on TGFBIp/βig-h3 protein and its functions in morphogenesis, extracellular matrix interactions, adhesion/migration, corneal dystrophy, tumorigenesis, angiogenesis, nephropathies, osteogenesis, wound healing and inflammation.     

A computational proposal for designing structured RNA pools for in vitro selection of RNAs

Although in vitro selection technology is a versatile experimental tool for discovering novel synthetic RNA molecules, finding complex RNA molecules is difficult because most RNAs identified from random sequence pools are simple motifs, consistent with recent computational analysis of such sequence pools. Thus, enriching in vitro selection pools with complex structures could increase the probability of discovering novel RNAs. Here we develop an approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a "mixing matrix" approach combined with a graph theory analysis. We define five classes of mixing matrices motivated by covariance mutations in RNA; these constructs define nucleotide transition rates and are applied to chosen starting sequences to yield specific nonrandom pools. We examine the coverage of sequence space as a function of the mixing matrix and starting sequence via clustering analysis. We show that, in contrast to random sequences, which are associated only with a local region of sequence space, our designed pools, including a structured pool for GTP aptamers, can target specific motifs. It follows that experimental synthesis of designed pools can benefit from using optimized starting sequences, mixing matrices, and pool fractions associated with each of our constructed pools as a guide. Automation of our approach could provide practical tools for pool design applications for in vitro selection of RNAs and related problems.     

Activation of peroxisome proliferator-activated receptor-gamma protects pancreatic beta-cells from cytokine-induced cytotoxicity via NF kappaB pathway

Diabetes mellitus is characterized by cytokine-induced insulitis and a deficit in beta-cell mass. Ligands for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) have been shown to have anti-inflammatory effects in various experimental models. We questioned whether activation of endogenous PPAR-gamma by either PPAR-gamma ligands or adenoviral-directed overexpression of PPAR-gamma (Ad-PPAR-gamma) could inhibit cytokine-induced beta-cell death in RINm5F (RIN) cells, a rat insulinoma cell line. Treatment of RIN cells with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) induced beta-cell damage through NF kappaB-dependent signaling pathways. Activation of PPAR-gamma by PPAR-gamma ligands or Ad-PPAR-gamma inhibited IL-1 beta and IFN-gamma-stimulated nuclear translocation of the p65 subunit and DNA binding activity. NF kappaB target gene expression and their product formation, namely inducible nitric oxide synthase and cyclooxygenase-2 were decreased by PPAR-gamma activation, as established by real-time PCR, Western blots and measurements of NO and PGE(2). The mechanism by which PPAR-gamma activation inhibited NF kappaB-dependent cell death signals appeared to involve the inhibition of I kappa B alpha degradation, evidenced by inhibition of cytokine-induced NF kappaB-dependent signaling events by Ad-I kappaB alpha (S32A, S36A), non-degradable I kappaB alpha mutant. I kappaB beta mutant, Ad-I kappaB beta (S19A, S23A) was not effective in preventing cytokine toxicity. Furthermore, a protective effect of PPAR-gamma ligands was proved by assaying for normal insulin secreting capacity in response to glucose in isolated rat pancreatic islets. The beta-cell protective function of PPAR-gamma ligands might serve to counteract cytokine-induced beta-cell destruction.