Induction of Endothelial Cell Differentiation in Vitro by Fibroblast-Derived Soluble Factors

Recent studies have suggested that fibroblasts, widely distributed mesenchymal cells, not only function to sustain various organs and tissues as stroma cells but also act directly to regulate adjacent cell behavior including migration, proliferation, and differentiation. Since fibroproliferative diseases and lesions (fibroplasia) are accompanied by new capillary growth (angiogenesis), we hypothesized that fibroblasts may have direct effects on endothelial cell behavior, independent of the elaboration of extracellular matrix, that are relevant to complex process of angiogenesis. To test this hypothesis, bovine aortic endothelial cells were cocultured in collagen gels with human skin fibroblasts. This coculture system caused the endothelial cells to become spindle shaped and to organize into a capillary-like structure within the collagen gels. We found that fibroblast-conditioned medium (FCM) also induced endothelial cells initially to elongate and subsequently to organize into a capillary-like structure within collagen gels. While FCM had no significant effect on endothelial cell DNA synthesis, the soluble factor(s) in FCM increased endothelial cell motility in an in vitro wound assay and in a Boyden chamber assay. The chemoattractant(s) in FCM was alkaline (pH 9.0)—and acid (pH 3.0)—stable, relatively heat stable (stable at 60°C for 30 min, unstable at 98°C for 3 min), dithiothreitol (DTT)-sensitive, and bound to an anionic exchange resin (DEAE-cellulose). Another factor(s) stimulated endothelial cell reorganization into capillary-like structure both within a collagen gel and on a reconstituted basement membrane matrix, Matrigel. This factor(s) was alkaline (pH 9.0)—and acid (pH 3.0)—stable, heat (98°C for 3 min)stable, and DTT-sensitive and bound an anionic exchange resin (DEAE-cellulose). These in vitro results suggest that fibroblasts secrete soluble factors that can influence endothelial cell behaviors relevant to the angiogenesis process with possible implications for vascularization in fibroproliferative conditions.     

Synthesis and structure based optimization of 2-(4-phenoxybenzoyl)-5-hydroxyindole as a novel CaMKII inhibitor

Based on 2-(4-phenoxybenzoyl)-5-hydroxyindole (2), a novel structural class of CaMKII inhibitors were synthesized and further optimized. The strong acidity of the hydroxyl group and the lipophilic group at the 4 and 6-positions were found to be necessary for strong CaMKII inhibition. Compound 25 was identified as a promising compound with 50-fold more potent inhibitory activity for CaMKII than 2. Compound 25 also showed high selectivity for CaMKII over off-target kinases.     

Taxonomic relationships of Ansonia anotis Inger, Tan, and Yambun, 2001 and Pedostibes maculatus (Mocquard, 1890), with a description of a new genus (Amphibia, Bufonidae)

Examination of types and recently collected specimens revealed that Ansonia anotis Inger, Tan, and Yambun, 2001 and Pedostibes maculatus (Mocquard, 1890), both described from Kinabalu, Sabah, Malaysia, are hardly differentiated morphologically. Analyses of a total of 2,427 bp of the 12S rRNA, tRNA(val), and 16S mitochondrial rRNA genes revealed that the two species are very close genetically. Thus A. anotis is regarded as conspecific and is synonymized with P. maculatus. Genetically, this species proved to form a lineage distinct from other bufonids from Southeast Asia, including species of Ansonia and Pedostibes. Because the species has also some unique morphological traits different from known bufonid genera, we propose to establish a new genus for Nectophryne maculata Mocquard, 1890.     

Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance

Prefoldin is a jellyfish-shaped hexameric chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. In this work, we characterized the organic solvent tolerance of Escherichia coli cells that overexpress prefoldin and group II chaperonin from a hyperthermophilic archeaum, Pyrococcus horikoshii OT3. The colony-forming efficiency of E. coli cells overexpressing prefoldin increased by 1,000-fold and decreased the accumulation of intracellular organic solvent. The effect was impaired by deletions of the region responsible for the chaperone function of prefoldin. Therefore, we concluded that prefoldin endows E. coli cells by preventing accumulation of intracellular organic solvent through its molecular chaperone activity.     

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14 ± 0.23 mM^− 1s^− 1, 4.54 ± 0.09 mM^− 1s^− 1, 0.087 ± 0.02 mM^− 1s^− 1 and 153.5 ± 7.1 mM^− 1s^− 1, respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.     

Duchenne and Becker muscular dystrophy: from gene diagnosis to molecular therapy

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked muscular dystrophies. The isolation of the defective gene in DMD/BMD has led to a better understanding of the disease process and has promoted studies regarding the application of molecular therapy. The purpose of this review is to present the progress made in this area of research with particular reference to dystrophin Kobe. Based on the results from the molecular analysis of dystrophin Kobe, we propose a novel molecular therapeutic method for DMD in which antisense oligonucleotides transform DMD into a milder phenotype by inducing exon skipping. In addition, current proposals for the molecular therapy of DMD are discussed.     

Interferon-Tau Attenuates Uptake of Nanoparticles and Secretion of Interleukin-1β in Macrophages

Background

Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1β, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages.

Methods and Results

IFNT dose-dependently inhibited IL-1β secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1β and mature IL-1β. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1β mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1β secretion was restored by anti-IL-10 neutralizing antibody.

Conclusions

Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1β secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1β induction. It may be a therapeutic alternative to IFNA and IFNB.     

In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5' splice sites

We have found that two previously reported exonic mutations in the PINK1 and PARK7 genes affect pre-mRNA splicing. To develop an algorithm to predict underestimated splicing consequences of exonic mutations at the 5′ splice site, we constructed and analyzed 31 minigenes carrying exonic splicing mutations and their derivatives. We also examined 189 249 U2-dependent 5′ splice sites of the entire human genome and found that a new variable, the SD-Score, which represents a common logarithm of the frequency of a specific 5′ splice site, efficiently predicts the splicing consequences of these minigenes. We also employed the information contents (R_i) to improve the prediction accuracy. We validated our algorithm by analyzing 32 additional minigenes as well as 179 previously reported splicing mutations. The SD-Score algorithm predicted aberrant splicings in 198 of 204 sites (sensitivity = 97.1%) and normal splicings in 36 of 38 sites (specificity = 94.7%). Simulation of all possible exonic mutations at positions −3, −2 and −1 of the 189 249 sites predicts that 37.8, 88.8 and 96.8% of these mutations would affect pre-mRNA splicing, respectively. We propose that the SD-Score algorithm is a practical tool to predict splicing consequences of mutations affecting the 5′ splice site.     

Nicotine affects mineralized nodule formation by the human osteosarcoma cell line Saos-2

Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. In the present study, we examined the effect of nicotine on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in the human osteosarcoma cell line Saos-2. The cells were cultured with Dulbecco's modified Eagle medium containing 10% fetal bovine serum with 0, 10(-4) M, and 10(-3) M nicotine for up to 14 days. Mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium E-test kit. The expression of extracellular matrix proteins was estimated by determining the levels of their mRNAs using the real-time polymerase chain reaction. Mineralized nodule formation and calcium content in mineralized nodules were remarkably suppressed by nicotine on days 10 and 14 of culture, respectively. ALPase activity as well as type I collagen and osteopontin expression also decreased in the presence of nicotine after 5, 10, and 14 days of culture, respectively. By contrast, the amount of bone sialoprotein increased during 14 days of culture with nicotine. These results suggest that nicotine suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.