Engineering of a Glycerol Utilization Pathway for Amino Acid Production by Corynebacterium glutamicum

The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures.     

IL-15 links TLR2/1-induced macrophage differentiation to the vitamin D-dependent antimicrobial pathway

An essential function of the innate immune system is to directly trigger antimicrobial mechanisms to defend against invading pathogens. In humans, one such pathway involves activation by TLR2/1L leading to the vitamin D-dependent induction of antimicrobial peptides. In this study, we found that TLR2/1-induced IL-15 was required for induction of CYP27b1, the VDR and the downstream antimicrobial peptide cathelicidin. Although both IL-15 and IL-4 triggered macrophage differentiation, only IL-15 was sufficient by itself to induce CYP27b1 and subsequent bioconversion of 25-hydroxyvitamin D3 (25D3) into bioactive 1,25D3, leading to VDR activation and induction of cathelicidin. Finally, IL-15-differentiated macrophages could be triggered by 25D3 to induce an antimicrobial activity against intracellular Mycobacterium tuberculosis. Therefore, IL-15 links TLR2/1-induced macrophage differentiation to the vitamin D-dependent antimicrobial pathway.     

Increased levels of circulating microparticles in primary Sj?gren's syndrome,systemic lupus erythematosus and rheumatoid arthritis and relation with disease activity

Introduction

Cell stimulation leads to the shedding of phosphatidylserine (PS)-rich microparticles (MPs). Because autoimmune diseases (AIDs) are characterized by cell activation, we investigated level of circulating MPs as a possible biomarker in primary Sjögren''s syndrome (pSS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).

Methods

We measured plasma levels of total, platelet and leukocyte MPs by prothrombinase capture assay and flow cytometry in 43 patients with pSS, 20 with SLE and 24 with RA and in 44 healthy controls (HCs). Secretory phospholipase A2 (sPLA2) activity was assessed by fluorometry. Soluble CD40 ligand (sCD40L) and soluble P-selectin (sCD62P), reflecting platelet activation, were measured by ELISA.

Results

Patients with pSS showed increased plasma level of total MPs (mean ± SEM 8.49 ± 1.14 nM PS equivalent (Eq), P < 0.0001), as did patients with RA (7.23 ± 1.05 n PS Eq, P = 0.004) and SLE (7.3 ± 1.25 nM PS Eq, P = 0.0004), as compared with HCs (4.13 ± 0.2 nM PS Eq). Patients with AIDs all showed increased level of platelet MPs (P < 0.0001), but only those with pSS showed increased level of leukocyte MPs (P < 0.0001). Results by capture assay and flow cytometry were correlated. In patients with high disease activity according to extra-glandular complications (pSS), DAS28 (RA) or SLEDAI (SLE) compared with low-activity patients, the MP level was only slightly increased in comparison with those having a low disease activity. Platelet MP level was inversely correlated with anti-DNA antibody level in SLE (r = -0.65; P = 0.003) and serum β2 microglobulin level in pSS (r = -0.37; P < 0.03). The levels of total and platelet MPs were inversely correlated with sPLA2 activity (r = -0.37, P = 0.0007; r = -0.36, P = 0.002, respectively). sCD40L and sCD62P concentrations were significantly higher in pSS than in HC (P ≤ 0.006).

Conclusions

Plasma MP level is elevated in pSS, as well as in SLE and RA, and could be used as a biomarker reflecting systemic cell activation. Level of leukocyte-derived MPs is increased in pSS only. The MP level is low in case of more severe AID, probably because of high secretory phospholipase A2 (sPLA2) activity, which leads to consumption of MPs. Increase of platelet-derived MPs, sCD40L and sCD62P, highlights platelet activation in pSS.     

Growth at elevated CO2 concentrations leads to modified profiles of secondary metabolites in tobacco cv. SamsunNN and to increased resistance against infection with potato virus Y

The effect of elevated CO2 concentrations on the levels of secondary metabolites was investigated in tobacco plants grown under two nitrogen supply (5 and 8 mM NH4NO3) and CO2 conditions (350 and 1000 p.p.m.) each. High CO2 resulted in a dramatic increase of phenylpropanoids in the leaves, including the major carbon-rich compound chlorogenic acid (CGA) and the coumarins scopolin and scopoletin at both nitrogen fertilizations. This was accompanied by increased PAL activity in leaves and roots, which was even higher at the lower nitrogen supply. Hardly any change was observed for the structural phenolic polymer lignin and the sesquiterpenoid capsidiol. In contrast, elevated CO2 led to clearly decreased levels of the main nitrogen-rich constituent nicotine at the lower N-supply (5 mM NH4NO3) but not when plants were grown at the higher N-supply (8 mM NH4NO3). Inoculation experiments with potato virus Y (PVY) were used to evaluate possible ecological consequences of elevated CO2. The titre of viral coat-protein was markedly reduced in leaves under these conditions at both nitrogen levels. Since PR-gene expression and free salicylic acid (SA) levels remained unchanged at elevated CO2, we suggest that the accumulation of phenylpropanoids, for example, the major compound CGA and the coumarins scopolin and scopoletin may result in an earlier confinement of the virus at high CO2. Based on our results two final conclusions emerge. First, elevated CO2 leads to a shift in secondary metabolite composition that is dependent on the availability of nitrogen. Second, changes in the pool of secondary metabolites have important consequences for plant-pathogen interactions as shown for PVY as a test organism.     

Novel proteins in emulsions using in vitro compartmentalization

IVC (in vitro compartmentalization) provides a complete cell-free approach for the production of novel targeted proteins. IVC uses aqueous droplets, which contain DNA and components for protein production, within water-in-oil emulsions. Recent advances in the composition and formation, as well as the detection, sorting and recovery, of the droplets enable the evolution of the encoded protein. Furthermore, IVC technology permits the step-wise addition of reagents into the droplets, making them suitable for high-throughput applications - where synthetic enzymes with substrate specificity are selected for catalytic activity, binding and regulation. In the broad field of in vitro display, developments such as the incorporation of unnatural amino acids and the production of cell toxic proteins expand the diverse spectrum of future applications for IVC.     

TransientAgrobacterium-mediated gene expression in theArabidopsis hydroponics root system for subcellular localization studies

To a great extent, the cellular compartmentalization and molecular interactions are indicative of the function of a protein. The development of simple and efficient tools for testing the subcellular location of proteins is indispensable to elucidate the function of genes in plants. In this report, we assessed the feasibility ofAgrobacterium-mediated transformation of hydroponically grown roots to follow intracellular targeting of proteins fused to green fluorescent protein (GFP). We developed a simple in planta assay for subcellular localization of proteins inArabidopsis roots via transient transformation and tested this method by expressing a GFP fusion of a known nuclear protein, IQD1. Visualization of transiently expressed GFP fusion proteins in roots by means of confocal microscopy is superior to the analysis of green tissues because the roots are virtually transparent and free of chlorophyll autofluorescence.     

MT1-MMP-dependent invasion is regulated by TI-VAMP/VAMP7

Proteolytic degradation of the extracellular matrix (ECM) is one intrinsic property of metastatic tumor cells to breach tissue barriers and to disseminate into different tissues. This process is initiated by the formation of invadopodia, which are actin-driven, finger-like membrane protrusions. Yet, little is known on how invadopodia are endowed with the functional machinery of proteolytic enzymes [1, 2]. The key protease MT1-MMP (membrane type 1-matrix metalloproteinase) confers proteolytic activity to invadopodia and thus invasion capacity of cancer cells [3-6]. Here, we report that MT1-MMP-dependent matrix degradation at invadopodia is regulated by the v-SNARE TI-VAMP/VAMP7, hence providing the molecular inventory mediating focal degradative activity of cancer cells. As observed by TIRF microscopy, MT1-MMP-mCherry and GFP-VAMP7 were simultaneously detected at proteolytic sites. Functional ablation of VAMP7 decreased the ability of breast cancer cells to degrade and invade in a MT1-MMP-dependent fashion. Moreover, the number of invadopodia was dramatically decreased in VAMP7- and MT1-MMP-depleted cells, indicative of a positive-feedback loop in which the protease as a cargo of VAMP7-targeted transport vesicles regulates maturation of invadopodia. Collectively, these data point to a specific role of VAMP7 in delivering MT1-MMP to sites of degradation, maintaining the functional machinery required for invasion.