Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli

λ-Red system-based recombinogenic engineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. Here, we report the use of a single selectable marker to enhance the usefulness of this approach. The strategy is to utilize the thymidylate synthase A (thyA) gene, which encodes an enzyme involved in the synthesis of thymidine 5′-triphosphate, for both positive and negative selection. With this approach, we successfully created point mutations in plasmid and bacterial artificial chromosome (BAC) DNA containing the mouse Col10a1 gene. The results showed that the thyA selection system is highly efficient and accurate, giving an average of >90% selection efficiency. This selection system produces DNA that is free from permanent integration of unwanted sequences, thus allowing unlimited rounds of modifications if required.     

Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages

Virulizin, a novel biological response modifier, has demonstrated significant antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. The significant role of macrophages and NK (Natural killer) cells was implicated in the antitumor mechanism of Virulizin where expansion as well as increased activity of macrophages and NK cells were observed in mice treated with Virulizin. Depletion of macrophages compromised Virulizin-induced NK1.1⁺ cell infiltration into xenografted tumors and was accompanied by reduced antitumor efficacy. In the present study, involvement of macrophages in NK cell activation was investigated further. We found that depletion of NK cells in CD-1 nude mice by anti-ASGM1 antibody significantly compromised the antitumor activity of Virulizin. Cytotoxicity of NK cells isolated from Virulizin-treated mice was enhanced against NK-sensitive YAC-1 cells and C8161 human melanoma cells, but not against NK-insensitive P815 cells. An increased level of IL-12 was observed in the serum of mice treated with Virulizin. IL-12 mRNA and protein levels were also increased in peritoneal macrophages isolated from Virulizin-treated mice. Moreover, Virulizin-induced cytotoxic activity of NK cells isolated from the spleen was abolished when an IL-12 neutralizing antibody was co-administered. In addition, depletion of macrophages in mice significantly impaired Virulizin-induced NK cell cytotoxicty. Taken together, the results suggest that Virulizin induces macrophage IL-12 production, which in turn stimulates NK cell-mediated antitumor activity.     

Tumor necrosis factor-alpha stimulates gastric epithelial cell proliferation

TNF-alpha is a cytokine produced during gastric mucosal injury. We examined whether TNF-alpha could promote mucosal repair by stimulation of epithelial cell proliferation and explored further the underlying mechanisms in a rat gastric mucosal epithelial cell line (RGM-1). TNF-alpha treatment (1-10 ng/ml) for 12 or 24 h significantly increased cell proliferation but did not induce apoptosis in RGM-1 cells. TNF-alpha treatment significantly increased cytosolic phospholipase A(2) and cyclooxygenase-2 (COX-2) protein expression and PGE(2) level but did not affect the protein levels of EGF, basic fibroblast growth factor, and COX-1 in RGM-1 cells. The mRNA of TNF receptor (TNF-R) 2 but not of TNF-R1 was also increased. Dexamethasone dose dependently inhibited the stimulatory effect of TNF-alpha on cell proliferation, which was associated with a significant decrease in cellular COX-2 expression and PGE(2) level. A selective COX-2 inhibitor 3-(3-fluorophenyl)-4-[4-(methylsulfonyl)phenyl]-5,5-dimethyl-(5)H-furan-2-one (DFU) by itself had no effect on basal cell proliferation but significantly reduced the stimulatory effect of TNF-alpha on RMG-1 cells. Combination of dexamethasone and DFU did not produce an additive effect. PGE(2) significantly reversed the depressive action of dexamethasone on cell proliferation. These results suggest that TNF-alpha plays a regulatory role in epithelial cell repair in the gastric mucosa via the TNF-alpha receptor and activation of the arachidonic acid/PG pathway.     

NAD+ and metal-ion dependent hydrolysis by family 4 glycosidases: structural insight into specificity for phospho-beta-D-glucosides

The import of disaccharides by many bacteria is achieved through their simultaneous translocation and phosphorylation by the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS). The imported phospho-disaccharides are, in some cases, subsequently hydrolyzed by members of the unusual glycoside hydrolase family GH4. The GH4 enzymes, occasionally found also in bacteria such as Thermotoga maritima that do not utilise a PEP-PTS system, require both NAD(+) and Mn(2+) for catalysis. A further curiosity of this family is that closely related enzymes may show specificity for either alpha-d- or beta-d-glycosides. Here, we present, for the first time, the three-dimensional structure (using single-wavelength anomalous dispersion methods, harnessing extensive non-crystallographic symmetry) of the 6-phospho-beta-glycosidase, BglT, from T.maritima in native and complexed (NAD(+) and Glc6P) forms. Comparison of the active-center structure with that of the 6-phospho-alpha-glucosidase GlvA from Bacillus subtilis reveals a striking degree of structural similarity that, in light of previous kinetic isotope effect data, allows the postulation of a common reaction mechanism for both alpha and beta-glycosidases. Given that the "chemistry" occurs primarily on the glycone sugar and features no nucleophilic attack on the intact disaccharide substrate, modulation of anomeric specificity for alpha and beta-linkages is accommodated through comparatively minor structural changes.     

Neurofilament-light increases the cell surface expression of the N-methyl-D-aspartate receptor and prevents its ubiquitination

NMDA (N-methyl-D-aspartate) subtype of glutamate receptors are core components of dendritic spine postsynaptic densities (PSDs), in which they are anchored via their carboxy-terminal tails to cytoskeletal proteins. In this study, we examined the role of the neuronal intermediate filament protein, neurofilament-light (NF-L), also a component of the PSD, in the regulation of NMDA receptor (NMDAR) expression and function in a heterologous system. Coexpression of NF-L with NR1 or NR2B subunits of the NMDAR in HEK293 (human embryonic kidney 293) cells did not result in surface expression as measured by surface biotinylation and cell ELISAs, whereas the combined expression of the three elements resulted in a 20% increase in the surface abundance of NR1, along with a concomitant increase in NMDAR-mediated cytotoxicity. Investigating the origin of this increase, we found that the NR1 subunits are ubiquitinated in HEK293 cells, and that the coexpression of NF-L antagonizes this process. These results suggest a possible means of stabilization of NR1 via its association with NF-L.     

Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis

We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60) and 70 (Hsp70) of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.     

Direct visualization of asymmetric behavior in supported lipid bilayers at the gel-fluid phase transition

We utilize in situ, temperature-dependent atomic force microscopy to examine the gel-fluid phase transition behavior in supported phospholipid bilayers constructed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. The primary gel-fluid phase transition at T(m) occurs through development of anisotropic cracks in the gel phase, which develop into the fluid phase. At approximately 5 degrees C above T(m), atomic force microscopy studies reveal the presence of a secondary phase transition in all three bilayers studied. The secondary phase transition occurs as a consequence of decoupling between the two leaflets of the bilayer due to enhanced stabilization of the lower leaflet with either the support or the water entrained between the support and the bilayer. Addition of the transmembrane protein gramicidin A or construction of a highly defected gel phase results in elimination of this decoupling and removal of the secondary phase transition.