Mechanism of the ring contraction process in vitamin B12 biosynthesis by the anaerobe Propionibacterium shermanii under aerobic conditions

The mechanism of the ring contraction process during vitamin B(12) biosynthesis by the anaerobe Propionibacterium shermanii was investigated under both aerobic and anaerobic conditions by means of feeding experiments with delta-amino[1-(13)C]levulinic acid (a biosynthetic intermediate of tetrapyrrole) and delta-amino[1-(13)C,1,1,4-(18)O(3)]levulinic acid in combination with (13)C-NMR spectroscopy. We showed that the characteristic mechanism of the ring contraction process (the generation of precorrin-3x from formation of the gamma-lactone from the ring A acetate group at C1 and hydroxylation at C20 by molecular oxygen catalyzed by CobG, and the migration of ring D by cleavage of the carbon-oxygen bond at C1 of precorrin-3x) in the aerobe Pseudomonas denitrificans was not seen in P. shermanii under aerobic conditions, and the mechanism of the ring contraction process in P. shermanii was the same irrespective of the presence or absence of oxygen.     

A multipotent clonal human periodontal ligament cell line with neural crest cell phenotypes promotes neurocytic differentiation, migration, and survival

Repair of injured peripheral nerve is thought to play important roles in tissue homeostasis and regeneration. Recent experiments have demonstrated enhanced functional recovery of damaged neurons by some types of somatic stem cells. It remains unclear, however, if periodontal ligament (PDL) stem cells possess such functions. We recently developed a multipotent clonal human PDL cell line, termed cell line 1-17. Here, we investigated the effects of this cell line on neurocytic differentiation, migration, and survival. This cell line expressed the neural crest cell marker genes Slug, SOX10, Nestin, p75NTR, and CD49d and mesenchymal stem cell-related markers CD13, CD29, CD44, CD71, CD90, CD105, and CD166. Rat adrenal pheochromocytoma cells (PC12 cells) underwent neurocytic differentiation when co-cultured with cell line 1-17 or in conditioned medium from cell line 1-17 (1-17CM). ELISA analysis revealed that 1-17CM contained approximately 50 pg/ml nerve growth factor (NGF). Cell line 1-17-induced migration of PC12 cells, which was inhibited by a neutralizing antibody against NGF. Furthermore, 1-17CM exerted antiapoptotic effects on differentiated PC12 cells as evidenced by inhibition of neurite retraction, reduction in annexin V and caspase-3/7 staining, and induction of Bcl-2 and Bcl-xL mRNA expression. Thus, cell line 1-17 promoted neurocytic differentiation, migration, and survival through secretion of NGF and possibly synergistic factors. PDL stem cells may play a role in peripheral nerve reinnervation during PDL regeneration.     

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white

As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower ??1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.     

An ABC Transporter Mutation Alters Root Exudation of Phytochemicals That Provoke an Overhaul of Natural Soil Microbiota

Root exudates influence the surrounding soil microbial community, and recent evidence demonstrates the involvement of ATP-binding cassette (ABC) transporters in root secretion of phytochemicals. In this study, we examined effects of seven Arabidopsis (Arabidopsis thaliana) ABC transporter mutants on the microbial community in native soils. After two generations, only the Arabidopsis abcg30 (Atpdr2) mutant had significantly altered both the fungal and bacterial communities compared with the wild type using automated ribosomal intergenic spacer analysis. Similarly, root exudate profiles differed between the mutants; however, the largest variance from the wild type (Columbia-0) was observed in abcg30, which showed increased phenolics and decreased sugars. In support of this biochemical observation, whole-genome expression analyses of abcg30 roots revealed that some genes involved in biosynthesis and transport of secondary metabolites were up-regulated, while some sugar transporters were down-regulated compared with genome expression in wild-type roots. Microbial taxa associated with Columbia-0 and abcg30 cultured soils determined by pyrosequencing revealed that exudates from abcg30 cultivated a microbial community with a relatively greater abundance of potentially beneficial bacteria (i.e. plant-growth-promoting rhizobacteria and nitrogen fixers) and were specifically enriched in bacteria involved in heavy metal remediation. In summary, we report how a single gene mutation from a functional plant mutant influences the surrounding community of soil organisms, showing that genes are not only important for intrinsic plant physiology but also for the interactions with the surrounding community of organisms as well.The diversity of the microbial (bacterial and fungal) communities in soil is extraordinary; 1 g of soil contains more than 10 billion microorganisms belonging to thousands of different species (Roselló-Mora and Amann, 2001). Soil microbial populations are involved in a framework of interactions known to affect key environmental processes like biogeochemical cycling of nutrients, plant health, and soil quality (Pace, 1997; Barea et al., 2004; Giri et al., 2005). Most of the dynamic soil microbial interactions happen near the plant roots and root soil interface, an area called the rhizosphere (Lynch, 1990; Barea et al., 2002; Bais et al., 2006; Prithiviraj et al., 2007). Rhizosphere microbial communities differ between plant species (Priha et al., 1999; Innes et al., 2004; Batten et al., 2006), between ecotypes/chemotypes within species (Kowalchuk et al., 2006; Micallef et al., 2009), between different developmental stages of a given plant (Mougel et al., 2006; Weisskopf et al., 2006), and from those present in bulk soil (Broz et al., 2007). Different root types can also cultivate specific microbes (Lilijeroth et al., 1991; Yang and Crowley, 2000; Baudoin et al., 2002), a response that has generally been attributed to the microenvironments surrounding a root and the varying ability of specific root types to uptake nutrients from soils and secrete exudates. Recent evidence suggests that specific plant species support a highly coevolved soil fungal community, and this process is mediated by root-secreted compounds (Broeckling et al., 2008). Rhizosphere interactions are initiated by the release of compounds from different organisms, and it is believed that carbon compounds secreted by roots act as substrates for certain species of microbes in the rhizospshere (Morgan et al., 2005).Root exudates are released into the rhizosphere by three major pathways: diffusion, ion channel, and vesicle transport (Bertin et al., 2003). Recent evidence has implicated ATP-binding cassette (ABC) transporters in the secretion of phytochemicals present in the root exudates of Arabidopsis (Arabidopsis thaliana) and other plants (Loyola-Vargas et al., 2007; Sugiyama et al., 2007; Badri et al., 2008; Badri and Vivanco, 2009). ABC transporters are the largest family of membrane transport proteins found in all organisms from bacteria to humans (Higgins, 1992). These transmembrane proteins use the energy of ATP to pump a wide variety of substrates across the membranes, including peptides, carbohydrates, lipids, heavy metal chelates, inorganic acids, steroids, and xenobiotics (Goossens et al., 2003). ABC transporters are also involved in plant disease resistance at the leaf level (Kobae et al., 2006; Stein et al., 2006).There is accumulating evidence that root exudates play a role in establishing specific interactions with particular microbes in the rhizosphere (legume''s symbiotic interaction with rhizobia, interaction of plants with mycorrhizae, and plant-growth-promoting rhizobacteria [PGPR]; Nagahashi and Douds, 2000; Bais et al., 2006, 2008; Prithiviraj et al., 2007; Rudrappa et al., 2008). However, how root exudation processes that result in large-scale changes to the surrounding soil microbial community compared to individual microbes have not been determined, although some recent reviews have referred to it as a biological frontier (O''Connell et al., 1996; Kuiper et al., 2004; Ryan et al., 2009). In contrast, gene deletions and overexpression of specific genes in plants have been shown to attract or deter specific microbes (Widmer, 2007), herbivores, or their predators (Baldwin et al., 2006; Pandey and Baldwin, 2007; Mitra and Baldwin, 2008), and recently it has been shown that mutations in nonpigment floral chemistry genes affect flower visitation by native pollinators (Kessler et al., 2008). Thus, it is possible that gene expression manipulation leading to an altered spectrum of root exudates can influence the widespread community of soil organisms surrounding a plant. Using all available information described above, we present the most comprehensive study on the effect of a single gene mutation in an ABC transporter involved in root secretion of phytochemicals by Arabidopsis on the natural and coevolved soil microbial composition. We further determine the compounds that are likely to have an effect on moderating the microbial composition and characterized specific and natural microbes that interact with Arabidopsis in the soil by employing pyrosequencing technology.     

Effect of flavonoids on androgen and glucocorticoid receptors based on in vitro reporter gene assay

The effect of 32 flavonoids on androgen (AR) and glucocorticoid receptors (GR) was investigated using an MDA-kb2 human breast cancer cell line to predict potential AR and GR activities. Among them, 5-hydroxyflavone (7) had the highest AR antagonistic activity with an IC₅₀ value of 0.3 μM, whereas 6-methoxyflavone (11) had the highest induced luciferase activity with an EC₁₅₀ value of 0.7 μM. Genistein (2) and daizein (1) showed a sufficient increase of luciferase activities as their concentrations increased with EC₁₅₀ values of 4.4 and 10.1 μM, respectively. These findings provide evidence of a fundamental property of their structure–activity relationship with AR and/or GR.     

Discovery of selective PDE4B inhibitors

In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine derivatives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme. With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects in vivo and showed less emesis compared with Cilomilast.     

Comprehensive Analysis of Inflammatory Immune Mediators in Vitreoretinal Diseases

Inflammation affects the formation and the progression of various vitreoretinal diseases. We performed a comprehensive analysis of inflammatory immune mediators in the vitreous fluids from total of 345 patients with diabetic macular edema (DME, n = 92), proliferative diabetic retinopathy (PDR, n = 147), branch retinal vein occlusion (BRVO, n = 30), central retinal vein occlusion (CRVO, n = 13) and rhegmatogenous retinal detachment (RRD, n = 63). As a control, we selected a total of 83 patients with either idiopathic macular hole (MH) or idiopathic epiretinal membrane (ERM) that were free of major pathogenic intraocular changes, such as ischemic retina and proliferative membranes. The concentrations of 20 soluble factors (nine cytokines, six chemokines, and five growth factors) were measured simultaneously by multiplex bead analysis system. Out of 20 soluble factors, three factors: interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) were significantly elevated in all groups of vitreoretinal diseases (DME, PDR, BRVO, CRVO, and RRD) compared with control group. According to the correlation analysis in the individual patient''s level, these three factors that were simultaneously increased, did not show any independent upregulation in all the examined diseases. Vascular endothelial growth factor (VEGF) was significantly elevated in patients with PDR and CRVO. In PDR patients, the elevation of VEGF was significantly correlated with the three factors: IL-6, IL-8, and MCP-1, while no significant correlation was observed in CRVO patients. In conclusion, multiplex bead system enabled a comprehensive soluble factor analysis in vitreous fluid derived from variety of patients. Major three factors: IL-6, IL-8, and MCP-1 were strongly correlated with each other indicating a common pathway involved in inflammation process in vitreoretinal diseases.