Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.     

A dominant negative TAK1 inhibits cellular fibrotic responses induced by TGF-beta

Transforming growth factor-beta (TGF-beta) is crucially virulent in the progression of fibrotic disorders. TAK1 (TGF-beta activated kinase 1) is one of the mitogen-activated kinase kinase kinase (MAPKKK) that is involved in TGF-beta signal transduction. To elucidate the importance of TAK1 in TGF-beta-induced fibrotic marker expression, we investigated whether dominant negative TAK1 could suppress TGF-beta signaling. Based on the finding that TAB1 (TAK1 binding protein 1) binding to TAK1 is required for TAK1 activation, a minimal portion of TAK1 lacking kinase activity that binds to TAB1 was designed as a TAK1 dominant negative inhibitor (TAK1-DN). The effect of TAK1-DN was assessed in the cells that respond to TGF-beta stimulation and that lead to the increase in production of extracellular matrix (ECM) proteins. TAK1-DN, indeed, decreased the ECM protein production, indicating that TAK1-DN retains the ability to intercept the TGF-beta signaling effectively.     

Activation of hydrogen peroxide in horseradish peroxidase occurs within approximately 200 micro s observed by a new freeze-quench device

To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freeze-quench device with approximately 200 micro s of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H(2)O(2)). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at approximately 1 ms after the mixing of HRP and H(2)O(2) suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at approximately 200 micro s show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within approximately 200 micro s after mixing HRP and H(2)O(2). We conclude that the activation of H(2)O(2) in HRP at ambient temperature completes within approximately 200 micro s. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.     

Molecular, biochemical and immunological analyses of canine pancreatic DNase I

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.     

Occurrence of an alpha-galacturonosyl-ceramide in the dioxin-degrading bacterium Sphingomonas wittichii

The chemical structure of two glycosphingolipids (GSLs) found in the dioxin-degrading bacterium Sphingomonas wittichii strain RW1 was investigated by means of mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. One of the GSLs was alpha-D-glucuronosyl-ceramide, commonly present in Sphingomonas spp., and the other was proved to be alpha-D-galacturonosyl-ceramide, whose sugar configuration has not been reported before. In both GSLs the ceramide portion was composed of myristic acid or 2-hydroxy-myristic acid as the fatty acid, and 2-amino-1,3-octadecanediol or 2-amino-cis-13,14-methylene-1,3-eicosanediol as the dihydrosphingosine.     

Intraperitoneal immunization led to T cell hyporesponsiveness to Helicobacter pylori infection in mice

During Helicobacter pylori infection, T cell response is critical in the development of active gastritis and in protective immunity against infection. We studied gastric inflammation and T cell response in H. pylori-challenged mice following an intraperitoneal immunization, using whole H. pylori lysate (HpAg) in the absence of adjuvants. H. pylori-challenged mice without immunization developed moderate to severe gastric inflammation, and splenocytes from these mice produced Th1 polarizing cytokines in response to HpAg and Con A during the acute infection. On the other hand, immunized-challenged mice (those inoculated with H. pylori following immunization) had little or no gastric inflammation despite persistent H. pylori colonization. Our immunization primed splenocytes to produce IL-2, IFN-gamma, and IL-4 in response to HpAg and Con A before infection. However, these cells became hyporesponsive to both stimulants immediately after live bacterial challenge in terms of the production of these cytokines, especially IL-2 and IFN-gamma. CTLA-4 has been documented to be a negative regulator of IL-2 production and lymphoproliferation that induces peripheral tolerance and functions 24-72 hr after the initiation of T cell activation. Compared with challenged mice, T cells from immunized-challenged mice showed higher levels of CTLA-4 expression at 72 hr after oral challenge. These data suggested that our immunization inhibited the development of H. pylori-associated gastritis and induced T cell hyporesponsiveness to H. pylori infection, which might be mediated by the early induction of CTLA-4 following challenge.     

Whole chloroplast genome comparison of rice,maize, and wheat: implications for chloroplast gene diversification and phylogeny of cereals

The fully sequenced chloroplast genomes of maize (subfamily Panicoideae), rice (subfamily Bambusoideae), and wheat (subfamily Pooideae) provide the unique opportunity to investigate the evolution of chloroplast genes and genomes in the grass family (Poaceae) by whole-genome comparison. Analyses of nucleotide sequence variations in 106 cereal chloroplast genes with tobacco sequences as the outgroup suggested that (1) most of the genic regions of the chloroplast genomes of maize, rice, and wheat have evolved at similar rates; (2) RNA genes have highly conservative evolutionary rates relative to the other genes; (3) photosynthetic genes have been under strong purifying selection; (4) between the three cereals, 14 genes which account for about 28% of the genic region have evolved with heterogeneous nucleotide substitution rates; and (5) rice genes tend to have evolved more slowly than the others at loci where rate heterogeneity exists. Although the mechanism that underlies chloroplast gene diversification is complex, our analyses identified variation in nonsynonymous substitution rates as a genetic force that generates heterogeneity, which is evidence of selection in chloroplast gene diversification at the intrafamilial level. Phylogenetic trees constructed with the variable nucleotide sites of the chloroplast genes place maize basal to the rice-wheat clade, revealing a close relationship between the Bambusoideae and Pooideae.     

Evolutionary distance estimation under heterogeneous substitution pattern among lineages

Most of the sophisticated methods to estimate evolutionary divergence between DNA sequences assume that the two sequences have evolved with the same pattern of nucleotide substitution after their divergence from their most recent common ancestor (homogeneity assumption). If this assumption is violated, the evolutionary distance estimated will be biased, which may result in biased estimates of divergence times and substitution rates, and may lead to erroneous branching patterns in the inferred phylogenies. Here we present a simple modification for existing distance estimation methods to relax the assumption of the substitution pattern homogeneity among lineages when analyzing DNA and protein sequences. Results from computer simulations and empirical data analyses for human and mouse genes are presented to demonstrate that the proposed modification reduces the estimation bias considerably and that the modified method performs much better than the LogDet methods, which do not require the homogeneity assumption in estimating the number of substitutions per site. We also discuss the relationship of the substitution and mutation rate estimates when the substitution pattern is not the same in the lineages leading to the two sequences compared.