In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells

Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp-151, in a prokaryotic Escherichia coli expression system. Even though the E. coli-derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l-3,4-dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post-translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect-derived MAP contained converted dopa residues by in vivo post-translational modification. In addition, insect-derived MAP also had other post-translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post-translational modifications of MAP containing dopa and other modified amino acid residues.     

The Guts of Herbivorous Invertebrates: Promising Sources of Diverse Microorganisms with Unique Hemicellulolytic Systems

Invertebrates including insects are heterotrophic organisms and widely distributed in ecosystems. Due to their superior ability to digest various types of plant biomass taken as foods, some herbivorous invertebrates have attracted a great deal of industrial attention because such organisms include diverse cellulolytic and hemicellulolytic symbionts in their gut. Recent studies have shown that some of gut microorganisms of herbivores possess one or more extracellular fibrolytic enzymes with unique functions, which can be exploited as useful biocatalysts in various bioindustrial fields. Specifically, microbial hemicellulases with favorable biocatalytic activities are expected to be used for the development of excellent animal feed additives, production of prebiotics such as xylo‐ and mannooligosaccharides, and pretreatment of lignocellulosic biomass for the preparation of fermentable sugars. Here, we review our recent studies accomplished on several hemicellulolytic bacteria isolated from the guts of invertebrates and their glycoside hydrolases such as endo‐β‐1,4‐xylanases and endo‐β‐1,4‐mannanases.     

Enhancement of centelloside production from cultured plants of Centella asiatica by combination of thidiazuron and methyl jasmonate

In order to produce centellosides from whole plant cultures of Centella asiatica (L.) Urban, we evaluated the synergistic effects of thidiazuron (TDZ) and methyl jasmonate (MJ) on whole plant growth and centelloside production. After 4 weeks of treatment with 0.025 mg/L of TDZ coupled with 0.1 mM MJ, the production of madecassoside and asiaticoside from whole plant cultures was estimated to be 2.40- and 2.44-fold, respectively, above that of MJ elicitation alone. When whole plants were treated with a growth regulator and an elicitor, the growth of whole plants, as compared to the controls, did not differ. Additionally, total phytosyterol content in the leaves of whole plants co-treated with MJ and TDZ was 1.08-fold greater than those of MJ alone. These results demonstrate that combined treatments not only stimulate the accumulation of centellosides in the leaves but also inhibit the reduction of phytosterol levels caused by MJ elicitation.     

Enhancement of differentiation efficiency of hESCs into vascular lineage cells in hypoxia via a paracrine mechanism

Hypoxia is one way of inducing differentiation due to the activation of the key regulatory factor, Hypoxia-inducible factor 1 alpha (HIF-1α). However, the action of HIF-1α on the differentiation of hESCs was unclear until now. To investigate the effect of hypoxia on the differentiation of hESCs, we compared the differentiation efficacy into vascular lineage cells under normoxic and hypoxic conditions. We observed HIF-1α expression and the related expression of pro-angiogenic factors VEGF, bFGF, Ang-1 and PDGF in hEBs cultured under hypoxic conditions. Along with this, differentiation efficacy into vascular lineage cells was improved under hypoxic conditions. When HIF-1α was blocked by echinomycin, both angiogenic factors and the differentiation efficacy were down-regulated, suggesting that the enhancement of differentiation efficacy was caused by intrinsic up-regulation of HIF-1α and these pro-angiogenic factors under hypoxic condition. This response might be primarily regulated by the HIF-1α signal pathway, and hypoxia might be the key to improving the differentiation of hESCs into vascular lineage cells. Therefore, this study demonstrated that microenvironmental changes (i.e., hypoxia) can improve differentiation efficacy of hESCs into a vascular lineage without exogenous factors via cell-intrinsic up-regulation of angiogenic factors. These facts will contribute to the regulation of stem cell fate.     

Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.     

Relationship between active cervical range of motion and flexion-relaxation ratio in asymptomatic computer workers

A high prevalence and incidence of neck and shoulder pain is present in the working population, especially sedentary workers. Recent findings have indicated that the flexion-relaxation (FR) ratio in the cervical erector spinae (CES) muscles might be a significant criteria of neuromuscular impairment and function. Additionally, the active cervical range of motion (ROM) is frequently used for discriminating between individuals with pain and those who are asymptomatic. The purpose of the present study was to examine the relationship between the active cervical ROM and the FR ratio in a sample of regular visual display terminal (VDT) workers. In total, 20 asymptomatic male VDT workers were recruited. Active cervical ROM was measured by a cervical ROM (CROM) instrument. Surface electromyography (EMG) was used to collect myoelectrical signals from the CES muscles, and the FR ratio was calculated for statistical analysis. Pearson correlation coefficients were used to quantify the linear relationship between the active cervical ROM and the FR ratio. The values obtained for the FR ratio in the right CES muscles correlated significantly with the active cervical ROM measured in flexion (r=0.73, p<0.01), left lateral flexion (r=0.64, p<0.01), and left rotation (r=0.60, p<0.01). Flexion (r=0.74, p<0.01) and right lateral flexion (r=0.61, p<0.01) positively correlated with the left FR ratio. Extension and right rotation showed either a very weak or no correlation with the mean value of the right and left FR ratio. Our findings suggested that the cervical FR ratio had a positive correlation with cervical movements, and that changes of the activation patterns in CES demonstrated as cervical FR ratio are associated with reduction of the cervical range of motion including flexion and lateral flexion. In addition, muscular dysfunction of the CES could occur in regular computer workers prior to occurrence of pain; this means that the FR ratio could be used to evaluate the potential risk of neck discomfort in computer workers.     

Differential effects of peptidoglycan recognition proteins on experimental atopic and contact dermatitis mediated by Treg and Th17 cells

Skin protects the body from the environment and is an important component of the innate and adaptive immune systems. Atopic dermatitis and contact dermatitis are among the most frequent inflammatory skin diseases and are both determined by multigenic predisposition, environmental factors, and aberrant immune response. Peptidoglycan Recognition Proteins (Pglyrps) are expressed in the skin and we report here that they modulate sensitivity to experimentally-induced atopic dermatitis and contact dermatitis. Pglyrp3(-/-) and Pglyrp4(-/-) mice (but not Pglyrp2(-/-) mice) develop more severe oxazolone-induced atopic dermatitis than wild type (WT) mice. The common mechanism underlying this increased sensitivity of Pglyrp3(-/-) and Pglyrp4(-/-) mice to atopic dermatitis is reduced recruitment of Treg cells to the skin and enhanced production and activation Th17 cells in Pglyrp3(-/-) and Pglyrp4(-/-) mice, which results in more severe inflammation and keratinocyte proliferation. This mechanism is supported by decreased inflammation in Pglyrp3(-/-) mice following in vivo induction of Treg cells by vitamin D or after neutralization of IL-17. By contrast, Pglyrp1(-/-) mice develop less severe oxazolone-induced atopic dermatitis and also oxazolone-induced contact dermatitis than WT mice. Thus, Pglyrp3 and Pglyrp4 limit over-activation of Th17 cells by promoting accumulation of Treg cells at the site of chronic inflammation, which protects the skin from exaggerated inflammatory response to cell activators and allergens, whereas Pglyrp1 has an opposite pro-inflammatory effect in the skin.     

An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs

Background

Small RNAs generated by RNA polymerase IV (Pol IV) are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-si)RNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus.

Results

Paternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases.

Conclusions

Differential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2.     

Cloned, CD117 selected human amniotic fluid stem cells are capable of modulating the immune response

Amniotic fluid stem (AFS) cells are broadly multipotent, can be expanded extensively in culture, are not tumorigenic and can be readily cryopreserved for cell banking. Mesenchymal stem cells (MSC) show immunomodulatory activity and secrete a wide spectrum of cytokines and chemokines that suppress inflammatory responses, block mixed lymphocyte reactions (MLR) and other immune reactions, and have proven therapeutic against conditions such as graft-versus-host disease. AFS cells resemble MSCs in many respects including surface marker expression and differentiation potential. We therefore hypothesized that AFS cells may exhibit similar immunomodulatory capabilities. We present data to demonstrate that direct contact with AFS cells inhibits lymphocyte activation. In addition, we show that cell-free supernatants derived from AFS cells primed with total blood monocytes or IL-1β, a cytokine released by monocytes and essential in mediation of the inflammatory response, also inhibited lymphocyte activation. Further investigation of AFS cell-free supernatants by protein array revealed secretion of multiple factors in common with MSCs that are known to be involved in immune regulation including growth related oncogene (GRO) and monocyte chemotactic protein (MCP) family members as well as interleukin-6 (IL-6). AFS cells activated by PBMCs released several additional cytokines as compared to BM-MSCs, including macrophage inflammatory protein-3α (MIP-3α), MIP-1α and Activin. AFS cells also released higher levels of MCP-1 and lower levels of MCP-2 compared to BM-MSCs in response to IL-1β activation. This suggests that there may be some AFS-specific mechanisms of inhibition of lymphocyte activation. Our results indicate that AFS cells are able to suppress inflammatory responses in vitro and that soluble factors are an essential component in the communication between lymphocytes and AFS cells. Their extensive self-renewal capacity, possibility for banking and absence of tumorigenicity may make AFS cells a superior source of stable, well characterized "off the shelf" immunomodulatory cells for a variety of immunotherapies.     

Betacellulin-induced beta cell proliferation and regeneration is mediated by activation of ErbB-1 and ErbB-2 receptors

Background

Betacellulin (BTC), a member of the epidermal growth factor family, is known to play an important role in regulating growth and differentiation of pancreatic beta cells. Growth-promoting actions of BTC are mediated by epidermal growth factor receptors (ErbBs), namely ErbB-1, ErbB-2, ErbB-3 and ErbB-4; however, the exact mechanism for beta cell proliferation has not been elucidated. Therefore, we investigated which ErbBs are involved and some molecular mechanisms by which BTC regulates beta cell proliferation.

Methodology/Principal Findings

The expression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 mRNA was detected by RT-PCR in both a beta cell line (MIN-6 cells) and C57BL/6 mouse islets. Immunoprecipitation and western blotting analysis showed that BTC treatment of MIN-6 cells induced phosphorylation of only ErbB-1 and ErbB-2 among the four EGF receptors. BTC treatment resulted in DNA synthetic activity, cell cycle progression, and bromodeoxyuridine (BrdU)-positive staining. The proliferative effect was blocked by treatment with AG1478 or AG825, specific tyrosine kinase inhibitors of ErbB-1 and ErbB-2, respectively. BTC treatment increased mRNA and protein levels of insulin receptor substrate-2 (IRS-2), and this was blocked by the ErbB-1 and ErbB-2 inhibitors. Inhibition of IRS-2 by siRNA blocked cell cycle progression induced by BTC treatment. Streptozotocin-induced diabetic mice injected with a recombinant adenovirus expressing BTC and treated with AG1478 or AG825 showed reduced islet size, reduced numbers of BrdU-positive cells in the islets, and did not attain BTC-mediated remission of diabetes.

Conclusions/Significance

These results suggest that BTC exerts proliferative activity on beta cells through the activation of ErbB-1 and ErbB-2 receptors, which may increase IRS-2 expression, contributing to the regeneration of beta cells.