Identification and Characterization of a Novel aac(6′)-Iag Associated with the bla IMP-1–Integron in a Multidrug-Resistant Pseudomonas aeruginosa

In a continuing study from Dec 2006 to Apr 2008, we characterized nine multi-drug resistant Pseudomonas aeruginosa strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis of SpeI-digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-β-lactamase. PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated as In124, that carries an array of four gene cassettes: a novel aminoglycoside (AG) resistance gene, aac(6′)-Iag, bla _IMP-1, a truncated form of bla _IMP-1, and a truncated form of aac(6′)-Iag. The aac(6′)-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6′)-Iz. Recombinant AAC(6′)-Iag protein showed aminoglycoside 6′-N-acetyltransferase activity using thin-layer chromatography (TLC) and MS spectrometric analysis. Escherichia coli carrying aac(6′)-Iag showed resistance to amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not to gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for bla _IMP-1 and aac(6′)-Ig strongly suggested In124 is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually all AGs, suggesting that the In124 conjugal plasmid also possesses a gene conferring resistance to gentamicin.     

Matrix-Embedded Osteocytes Regulate Mobilization of Hematopoietic Stem/Progenitor Cells

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TNF-α Decreases VEGF Secretion in Highly Polarized RPE Cells but Increases It in Non-Polarized RPE Cells Related to Crosstalk between JNK and NF-κB Pathways

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.     

Wnt Signaling in Vertebrate Axis Specification

The Wnt pathway is a major embryonic signaling pathway that controls cell proliferation, cell fate, and body-axis determination in vertebrate embryos. Soon after egg fertilization, Wnt pathway components play a role in microtubule-dependent dorsoventral axis specification. Later in embryogenesis, another conserved function of the pathway is to specify the anteroposterior axis. The dual role of Wnt signaling in Xenopus and zebrafish embryos is regulated at different developmental stages by distinct sets of Wnt target genes. This review highlights recent progress in the discrimination of different signaling branches and the identification of specific pathway targets during vertebrate axial development.Wnt pathways play major roles in cell-fate specification, proliferation and differentiation, cell polarity, and morphogenesis (Clevers 2006; van Amerongen and Nusse 2009). Signaling is initiated in the responding cell by the interaction of Wnt ligands with different receptors and coreceptors, including Frizzled, LRP5/6, ROR1/2, RYK, PTK7, and proteoglycans (Angers and Moon 2009; Kikuchi et al. 2009; MacDonald et al. 2009). Receptor activation is accompanied by the phosphorylation of Dishev-elled (Yanagawa et al. 1995), which appears to transduce the signal to both the cell membrane and the nucleus (Cliffe et al. 2003; Itoh et al. 2005; Bilic et al. 2007). Another common pathway component is β-catenin, an abundant component of adherens junctions (Nelson and Nusse 2004; Grigoryan et al. 2008). In response to signaling, β-catenin associates with T-cell factors (TCFs) and translocates to the nucleus to stimulate Wnt target gene expression (Behrens et al. 1996; Huber et al. 1996; Molenaar et al. 1996).This β-catenin-dependent activation of specific genes is often referred to as the “canonical” pathway. In the absence of Wnt signaling, β-catenin is destroyed by the protein complex that includes Axin, GSK3, and the tumor suppressor APC (Clevers 2006; MacDonald et al. 2009). Wnt proteins, such as Wnt1, Wnt3, and Wnt8, stimulate Frizzled and LRP5/6 receptors to inactivate this β-catenin destruction complex, and, at the same time, trigger the phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2 (HIPK2) (Hikasa et al. 2010; Hikasa and Sokol 2011). Both β-catenin stabilization and the regulation of TCF protein function by phosphorylation appear to represent general strategies that are conserved in multiple systems (Sokol 2011). Thus, the signaling pathway consists of two branches that together regulate target gene expression (Fig. 1).Open in a separate windowFigure 1.Conserved Wnt pathway branches and components. In the absence of Wnt signals, glycogen synthase kinase 3 (GSK3) binds Axin and APC to form the β-catenin destruction complex. Some Wnt proteins, such as Wnt8 and Wnt3a, stimulate Frizzled and LRP5/6 receptors to inhibit GSK3 activity and stabilize β-catenin (β-cat). Stabilized β-cat forms a complex with T-cell factors (e.g., TCF1/LEF1) to activate target genes. Moreover, GSK3 inhibition leads to target gene derepression by promoting TCF3 phosphorylation by homeodomain-interacting protein kinase 2 (HIPK2) through an unknown mechanism, for which β-catenin is required as a scaffold. This phosphorylation results in TCF3 removal from target promoters and gene activation. Other Wnt proteins, such as Wnt5a and Wnt11, use distinct receptors such as ROR2 and RYK, in addition to Frizzled, to control the the cytoskeletal organization through core planar cell polarity (PCP) proteins, small GTPases (Rho/Rac/Cdc42), and c-Jun amino-terminal kinase (JNK).Other Wnt proteins, such as Wnt5a or Wnt11, strongly affect the cytoskeletal organization and morphogenesis without stabilizing β-catenin (Torres et al. 1996; Angers and Moon 2009; Wu and Mlodzik 2009). These “noncanonical” ligands do not influence TCF3 phosphorylation (Hikasa and Sokol 2011), but may use distinct receptors such as ROR1/2 and RYK instead of or in addition to Frizzled (Hikasa et al. 2002; Lu et al. 2004; Mikels and Nusse 2006; Nishita et al. 2006, 2010; Schambony and Wedlich 2007; Grumolato et al. 2010; Lin et al. 2010; Gao et al. 2011). In such cases, signaling mechanisms are likely to include planar cell polarity (PCP) components, such as Vangl2, Flamingo, Prickle, Diversin, Rho GTPases, and c-Jun amino-terminal kinases (JNKs), which do not directly affect β-catenin stability (Fig. 1) (Sokol 2000; Schwarz-Romond et al. 2002; Schambony and Wedlich 2007; Komiya and Habas 2008; Axelrod 2009; Itoh et al. 2009; Tada and Kai 2009; Sato et al. 2010; Gao et al. 2011). This simplistic dichotomy of the Wnt pathway does not preclude some Wnt ligands from using both β-catenin-dependent and -independent routes in a context-specific manner.Despite the existence of many pathway branches, only the β-catenin-dependent branch has been implicated in body-axis specification. Recent experiments in lower vertebrates have identified additional pathway components and targets and provided new insights into the underlying mechanisms.     

The Role of Individual Domains and the Significance of Shedding of ATP6AP2/(pro)renin Receptor in Vacuolar H+-ATPase Biogenesis

The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H⁺-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.     

Development and characterization of ten novel microsatellite loci for the emu (Dromaius novaehollandiae) and genetic diversity of Japanese farm populations

Molecular Biology Reports - The emu (Dromaius novaehollandiae) is a useful poultry animal farmed for fat, meat, and eggs. Genetic structure and relationships among farmed emu populations in Japan...     

Molecular characterization of rotaviruses obtained from patients with rotavirus-associated encephalitis/encephalopathy

Group A rotavirus (RVA) rarely causes severe complications such as encephalitis/encephalopathy. However, the pathophysiology of this specific complication remains unclear. Next-generation sequence analysis was used to compare the entire genome sequences of RVAs detected in patients with encephalitis/encephalopathy and gastroenteritis. This study enrolled eight patients with RVA encephalitis/encephalopathy and 10 with RVA gastroenteritis who were treated between February 2013 and July 2014. Viral RNAs were extracted from patients' stool, and whole-genome sequencing analysis was carried out to identify the specific gene mutations in RVA obtained from patients with severe neurological complications. Among the eight encephalitis/encephalopathy cases, six strains were DS-1-like G1P[8] and the remaining two were Wa-like G1P[8] (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). Meanwhile, eight of the 10 viruses detected in rotavirus gastroenteritis patients were DS-1-like G1P[8], and the remaining two were Wa-like G1P[8]. These strains were further characterized by conducting phylogenetic analysis. No specific clustering was demonstrated in RVAs detected from encephalitis/encephalopathy patients. Although the DS-1-like G1P[8] strain was predominant in both groups, no specific molecular characteristics were detected in RVAs from patients with severe central nervous system complications.     

Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.     

Treatment with β2‐Adrenoceptor Agonist in Vivo Induces Human Clock Gene,Per1, mRNA Expression in Peripheral Blood

This study examined whether in vivo exposure to a β₂‐adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real‐time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.