Large-Scale Engineering of the Corynebacterium glutamicum Genome

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a total of 250 kb (7.5% of the genome), the 11 genomic regions were loaded with cryptic prophages, transposons, and genes of unknown function which were dispensable for cell growth, indicating recent horizontal acquisitions to the genome. This provides an interesting background for functional genomic studies and can be used in the improvement of cell traits.     

A familial case of visceral toxocariasis due to consumption of raw bovine liver

We present 3 adult cases of visceral toxocariasis from the same family, who each consumed thin slices of raw bovine liver weekly, and developed eosinophilia and multiple small lesions in their livers and lungs. Serological examinations using the larval excretory–secretory product of Toxocara canis strongly indicated infection with Toxocara species larvae. The patients responded well to treatment with albendazole. Ingestion of raw liver from paratenic animals is considered to be a common transmission route of human toxocariasis, especially in adults.     

Acute lung inflammation and ventilator-induced lung injury caused by ATP via the P2Y receptors: an experimental study

Background

Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior.

Methods

The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG.

Results

We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin.

Conclusion

Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.     

Characterization of the {alpha}-helix region in domain 3 of the haemolytic lectin CEL-III: implications for self-oligomerization and haemolytic processes

CEL-III is a haemolytic lectin, which has two beta-trefoil domains (domains 1 and 2) and a beta-sheet-rich domain (domain 3). In domain 3 (residues 284-432), there is a hydrophobic region containing two alpha-helices (H8 and H9, residues 317-357) and a loop between them, in which alternate hydrophobic residues, especially Val residues, are present. To elucidate the role of the alpha-helix region in the haemolytic process, peptides corresponding to different parts of this region were synthesized and characterized. The peptides containing the sequence that corresponded to the loop and second alpha-helix (H9) showed the strongest antibacterial activity for Staphylococcus aureus and Bacillus subtilis through a marked permeabilization of the bacterial cell membrane. The recombinant glutathione S-transferase (GST)-fusion proteins containing domain 3 or the alpha-helix region peptide formed self-oligomers, whereas mutations in the alternate Val residues in the alpha-helix region lead to decreased oligomerization ability of the fusion proteins. These results suggest that the alpha-helix region, particularly its alternate Val residues are important for oligomerization of CEL-III in target cell membranes, which is also required for a subsequent haemolytic action.     

Phospholipase D from Streptoverticillium cinnamoneum: protein engineering and application for phospholipid production

This review is focusing on an industrially important enzyme, phospholipase D (PLD), exhibiting both transphosphatidylation and hydrolytic activities for various phospholipids. The transphosphatidylation activity of PLD is particularly useful for converting phosphatidylcholine (PC) into other phospholipids. During the last decade, the genes coding for PLD have been identified from various species including mammals, plants, yeast, and bacteria. However, detailed basic and applied enzymological studies on PLD have been hampered by the low productivity in these organisms. Efficient production of a recombinant PLD has also been unsuccessful so far. We recently isolated and characterized the PLD gene from Streptoverticillium cinnamoneum, producing a secretory PLD. Furthermore, we constructed an overexpression system for the secretory enzyme in an active and soluble form using Streptomyces lividans as a host for transformation of the PLD gene. The Stv. cinnamoneum PLD was proven to be useful for the continuous and efficient production of phosphatidylethanolamine (PE) from phosphatidylcholine. Thus, the secretory PLD is a promising catalyst for synthesizing new phospholipids possessing various polar head groups that show versatile physiological functions and may be utilized in food and pharmaceutical industries.     

Effects of flexible bridging ligands on DNA-binding of dinuclear ruthenium(II)-2,2′-bipyridine complexes

For reactions of [{RuCl(bpy)₂}₂(μ-BL)]²⁺ (bpy = 2,2′-bipyridine, BL = H₂N(CH₂)_nNH₂ (n = 4-8, 12), [Ru₂-BL]²⁺) with mononucleotides, the MLCT absorption bands of [Ru₂-BL]²⁺ blue-shifted with hyperchromism for GMP and hypochromism for TMP with time. Reactions of [Ru₂-BL]²⁺ with GMP or TMP proceed via initial Cl⁻ ions replacement by coordination to N7 of GMP and N3 of TMP, respectively. In competition binding experiments for [Ru₂-BL]²⁺ with GMP versus TMP, only GMP selectively coordinated to ruthenium(II). For reactions with calf thymus (CT) DNA, [Ru₂-BL]²⁺ complexes selectively bind to guanine residues of DNA. The higher degrees of binding of [Ru₂-BL]²⁺ to CT-DNA were observed with increasing n values for H₂N(CH₂)_nNH₂, which may be explained by the length of the bridging ligands. Studies on the inhibition of the restriction enzyme Acc I revealed that [Ru₂-BL]²⁺ complexes appear to be covalently favorable for the type of difunctional binding. In addition, it is very interesting to observe that circular dichroism spectroscopy of the supernatants obtained following the reactions of CT-DNA with racemic [Ru₂-BL]²⁺ show enrichments of the solutions in the ΔΔ isomers, demonstrating preferences of the ΛΛ isomers for covalent binding to CT-DNA.     

Identification and biochemical analysis of a homolog of a sulfate transporter from a vanadium-rich ascidian Ascidia sydneiensis samea

Background

Several species of ascidians accumulate extremely high levels of vanadium ions in the vacuoles of their blood cells (vanadocytes). The vacuoles of vanadocytes also contain many protons and sulfate ions. To maintain the concentration of sulfate ions, an active transporter must exist in the blood cells, but no such transporter has been reported in vanadium-accumulating ascidians.

Methods

We determined the concentration of vanadium and sulfate ions in the blood cells (except for the giant cells) of Ascidia sydneiensis samea. We cloned cDNA for an Slc13-type sulfate transporter, AsSUL1, expressed in the vanadocytes of A. sydneiensis samea. The synthetic mRNA of AsSUL1 was introduced into Xenopus oocytes, and its ability to transport sulfate ions was analyzed.

Results

The concentrations of vanadium and sulfate ions in the blood cells (except for the giant cells) were 38 mM and 86 mM, respectively. The concentration of sulfate ions in the blood plasma was 25 mM. The transport activity of AsSUL1 was dependent on sodium ions, and its maximum velocity and apparent affinity were 2500 pmol/oocyte/h and 1.75 mM, respectively.

General significance

This could account for active uptake of sulfate ions from blood plasma where sulfate concentration is 25 mM, as determined in this study.     

Development of continuous flow type hydrothermal reactor for hemicellulose fraction recovery from corncob

The semi-pilot scale of continuous flow type hydrothermal reactor has been investigated to separate hemicellulose fraction from corncob. We obtained the effective recovery of hemicellulose using tubular type reactor at 200 °C for 10 min. From constituent sugar analysis of corncob, 82.2% of xylan fraction was recovered as mixture of xylose, xylooligosaccharides and higher-xylooligosaccharide which has more than DP 10. During purification of solubilized fraction by hydrothermal reaction such as ultrafiltration and ion exchange resin, higher-xylooligosaccharide was recovered as the precipitate. This precipitate was identified as non-blanched xylan fraction which has from DP 11 to DP 21 mainly. In this system, only a small amount of furfural has been generated. This tubular reactor has a characteristic controllability of thermal history, and seems to be effective for sugar recovery from soft biomass like corncob.     

MGL2+ Dermal Dendritic Cells Are Sufficient to Initiate Contact Hypersensitivity In Vivo

Background

Dendritic cells (DCs) are the most potent antigen-presenting cells in the mammalian immune system. In the skin, epidermal Langerhans cells (LCs) and dermal dendritic cells (DDCs) survey for invasive pathogens and present antigens to T cells after migration to the cutaneous lymph nodes (LNs). So far, functional and phenotypic differences between these two DC subsets remain unclear due to lack of markers to identify DDCs.

Methodology/Principal Findings

In the present report, we demonstrated that macrophage galactose-type C-type lectin (MGL) 2 was exclusively expressed in the DDC subset in the skin-to-LN immune system. In the skin, MGL2 was expressed on the majority (about 88%) of MHCII⁺CD11c⁺ cells in the dermis. In the cutaneous LN, MGL2 expression was restricted to B220⁻CD8α^loCD11b⁺CD11c⁺MHCII^hi tissue-derived DC. MGL2⁺DDC migrated from the dermis into the draining LNs within 24 h after skin sensitization with FITC. Distinct from LCs, MGL2⁺DDCs localized near the high endothelial venules in the outer T cell cortex. In FITC-induced contact hypersensitivity (CHS), adoptive transfer of FITC⁺MGL2⁺DDCs, but not FITC⁺MGL2⁻DCs into naive mice resulted in the induction of FITC-specific ear swelling, indicating that DDCs played a key role in initiation of immune responses in the skin.

Conclusions/Significance

These results demonstrated the availability of MGL2 as a novel marker for DDCs and suggested the contribution of MGL2⁺ DDCs for initiating CHS.