Rapid detection of blaIMP-6 by amplification refractory mutation system

Klebsiella pneumoniae resistant to almost all ß-lactams except imipenem designated as ISMRK (imipenem-susceptible meropenem-resistant Klebsiella) is emerging in Japan. All ISMRK carries bla_IMP-6 which differs from bla_IMP-1 by only a single nucleotide at position 640. We devised a rapid detection system of bla_IMP-6 by using ARMS PCR.     

Chk1 phosphorylates the tumour suppressor Mig-6, regulating the activation of EGF signalling

The tumour suppressor gene product Mig-6 acts as an inhibitor of epidermal growth factor (EGF) signalling. However, its posttranslational modifications and regulatory mechanisms have not been elucidated. Here, we investigated the phosphorylation of human Mig-6 and found that Chk1 phosphorylated Mig-6 in vivo as well as in vitro. Moreover, EGF stimulation promoted phosphorylation of Mig-6 without DNA damage and the phosphorylation was inhibited by depletion of Chk1. EGF also increased Ser280-phosphorylated Chk1, a cytoplasmic-tethering form, via PI3K pathway. Mass spectrometric analyses suggested that Ser 251 of Mig-6 was a major phosphorylation site by Chk1 in vitro and in vivo. Substitution of Ser 251 to alanine increased inhibitory activity of Mig-6 against EGF receptor (EGFR) activation. Moreover, EGF-dependent activation of EGFR and cell growth were inhibited by Chk1 depletion, and were rescued by co-depletion of Mig-6. Our results suggest that Chk1 phosphorylates Mig-6 on Ser 251, resulting in the inhibition of Mig-6, and that Chk1 acts as a positive regulator of EGF signalling. This is a novel function of Chk1.     

The planarian P2X homolog in the regulation of asexual reproduction

The growth in size of freshwater planarians in response to nutrient intake is limited by the eventual separation of tail and body fragments in a process called fission. The resulting tail fragment regenerates the entire body as an artificially amputated tail fragment would do, and the body fragment regenerates a tail, resulting in two whole planarians. This regenerative ability is supported by pluripotent somatic stem cells, called neoblasts, which are distributed throughout almost the entire body of the planarian. Neoblasts are the only planarian cells with the ability to continuously proliferate and give rise to all types of cells during regeneration, asexual reproduction, homeostasis, and growth. In order to investigate the molecular characteristics of neoblasts, we conducted an extensive search for neoblast-specific genes using the High Coverage Expression Profiling (HiCEP) method, and tested the function of the resulting candidates by RNAi. Disruption of the expression of one candidate gene, DjP2X-A (Dugesia japonica membrane protein P2X homologue), resulted in a unique phenotype. DjP2X-A RNAi leads to an increase of fission events upon feeding. We confirmed by immunohistochemistry that DjP2X-A is a membrane protein, and elucidated its role in regulating neoblast proliferation, thereby explaining its unique phenotype. We found that DjP2X-A decreases the burst of neoblast proliferation that normally occurs after feeding. We also found that DjP2X-A is required for normal proliferation in starved animals. We propose that DjP2X-A modulates stem cell proliferation in response to the nutritional condition.     

Klotho coreceptors inhibit signaling by paracrine fibroblast growth factor 8 subfamily ligands

It has been recently established that Klotho coreceptors associate with fibroblast growth factor (FGF) receptor tyrosine kinases (FGFRs) to enable signaling by endocrine-acting FGFs. However, the molecular interactions leading to FGF-FGFR-Klotho ternary complex formation remain incompletely understood. Here, we show that in contrast to αKlotho, βKlotho binds its cognate endocrine FGF ligand (FGF19 or FGF21) and FGFR independently through two distinct binding sites. FGF19 and FGF21 use their respective C-terminal tails to bind to a common binding site on βKlotho. Importantly, we also show that Klotho coreceptors engage a conserved hydrophobic groove in the immunoglobulin-like domain III (D3) of the "c" splice isoform of FGFR. Intriguingly, this hydrophobic groove is also used by ligands of the paracrine-acting FGF8 subfamily for receptor binding. Based on this binding site overlap, we conclude that while Klotho coreceptors enhance binding affinity of FGFR for endocrine FGFs, they actively suppress binding of FGF8 subfamily ligands to FGFR.     

Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for Thraustochytrids

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo(r)), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo(r) marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo(r) mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C(20:3n-6)) and eicosatetraenoic acid (C(20:4n-3)), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C(20:4n-6)) and eicosapentaenoic acid (C(20:5n-3)), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.     

Construction of multi-functional extracellular matrix proteins that inhibits migration and tube formation of endothelial cells

Artificial extracellular matrix (ECM) proteins have been designed that have strong cell-adhesive activity and active functional units that inhibit network formation among vascular endothelial cells. A laminin-derived sequence (YIGSR) that blocks migration of vascular endothelial cells was designed to incorporate into an elastin-derived structural unit. The designed ECM fusion protein also had a cell-adhesive RGDN sequence that conferred an intense migration-inhibitory effect. The resultant ECM showed cell-adhesive activity superior to that of the synthetic YIGSR peptide and it blocked the migration of vascular endothelial cells. Furthermore, the designed ECM inhibited the angiogenic activity of a collagen gel. The engineering strategy of designing multi-functional ECM proteins could be applied to support novel tissue engineering techniques.     

Mycoplasma mobile cells elongated by detergent and their pivoting movements in gliding

Mycoplasma mobile glides on solid surfaces by the repeated binding of leg structures to sialylated oligosaccharide fixed on a solid surface. To obtain information about the propulsion caused by the leg, we made elongated and stiff cells using a detergent. Within 30 min after the cells were treated with 0.1% Tween 60, the cells were elongated from 0.8 μm to 2.2 μm in length while maintaining their gliding activity. Fluorescence and electron microscopy showed that a part of the cytoskeletal structure was elongated, while the localization of proteins involved in the gliding was not modified significantly. The elongated cells glided with repeated pivoting around the cellular position of gliding machinery by 10 degrees of amplitude at a frequency of 2 to 3 times per second, suggesting that the propulsion in a line perpendicular to the cell axis can occur with different timings. The pivoting speed decreased as the cell length increased, probably from the load generated by the friction. The torque required to achieve the actual pivoting increased with the cell length without saturation, reaching 54.7 pN nm at 4.3 μm in cell length.     

Facile synthesis of 4-O-β-N-Acetylchitooligosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone based on the transformation of chitooligosaccharide and its suppressive effects against the furylfuramide-induced SOS response

A facile synthesis method is described for transforming the reducing-end residue of chitooligosaccharides (DP 2-4) into lactone. The desired 4-O-β-N-acetylchitooligosyl lactones (GN(n)L) were conveniently prepared from chitooligosaccharides by consecutive dehydration and oxidation reactions to afford 4-O-β-tri-N-acetylchitotriosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN(3)L), 4-O-β-di-N-acetylchitobiosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN(2)L), and 4-O-β-2-acetamido-2-deoxy-D-glucopyranosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GNL). The resulting lactone derivatives exhibited considerable suppression (42.6-54.3% at a concentration of 400 μM) in umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2). Lactonization of the chitooligosaccharides was found to be essential for their suppression of the SOS-inducing activity.