Takashi Suematsu

Multidrug Efflux Systems Play an Important Role in the Invasiveness of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic human pathogen. Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice. Four multidrug efflux systems have been described in P. aeruginosa, however, their contribution to virulence is unclear. To clarify the role of efflux systems in invasiveness, P. aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia. All efflux mutants except a ΔmexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a ΔmexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001). The other mutants, including ΔmexB and ΔmexXY mutants, were intermediate between WT and the ΔmexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the ΔmexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT. We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.

Anti-Clostridium difficile Potential of Tetramic Acid Derivatives from Pseudomonas aeruginosa Quorum-Sensing Autoinducers

ABSTRACT We have examined the potential bactericidal activities of several tetramic acids derived from Pseudomonas autoinducers against Clostridium difficile, a cause of antibiotic-associated pseudomembranous colitis. Clinical isolates of C. difficile (n = 4) were incubated in broth with a chemically synthesized Pseudomonas autoinducer and its tetramic acid derivatives. The structure-activity relationship and the mechanisms of action were examined by a time-killing assay and by determination of the morphological/staining characteristics. The use of some tetramic acids derived from N-3-oxododecanoyl l-homoserine lactone resulted in more than 3-log reductions in the viability of C. difficile within 30 min at 30 μM. The outer membrane was suggested to be one of the targets for the bactericidal activity of tetramic acid, because disturbance of the bacterial outer surface was demonstrated by alteration of the Gram-staining characteristic and electron microscopy. The data for the tetramic acid derivatives demonstrate that the keto-enol structure and the length of the acyl side chain of tetramic acid may be essential for the antibacterial activity of this molecule. These results suggest the potential for tetramic acid derivatives to be novel agents with activity against C. difficile.

Distribution of acetylcholinesterase activity in the rat embryonic heart with reference to HNK-1 immunoreactivity in the conduction tissue

Acetylcholinesterase (AChE) activity was topographically investigated in the presumptive cardiac conduction tissue regions visualized by HNK-1 immunoreactivity in rat embryos, and AChE-positive cells were examined with the electron microscope. On embryonic day (ED) 14.5, when HNK-1 was most intensely visualized, AChE activity could not be detected enzyme-histochemically in the conduction tissue regions, except in the ventricular trabeculae and part of the AV node. On ED 16.5, however, the AChE activity was clearly demonstrated in some parts of the developing conduction tissue. One exception was the AV node region, where an AChE-positive area was in close proximity to an area showing HNK-1 immunoreactivity but did not overlap. Furthermore, AChE activity was demonstrated predominantly in the ventricular trabeculae, including cardiac myocytes, but was rather weak in the atrium. With the electron microscope, AChE reaction products were observed predominantly intracellulary in both developing conduction tissue cells and developing ordinary myocytes, and no reactivity was found in neuronal components. From ED 18.5 until birth, both AChE activity and HNK-1 immunoreactivity faded away in the conduction tissue. Thus, transient AChE activity in the embryonic heart seems to be different from the developing adult form and may be related to a morphogenetic function in embryonic tissues, as proposed by other authors.

MAGNIFYING ENDOSCOPIC OBSERVATION WITH NARROW BAND IMAGING FOR SPECIALIZED INTESTINAL METAPLASIA IN BARRETT'S ESOPHAGUS WITH SPECIAL REFERENCE TO LIGHT BLUE CRESTS

Aim:  Barretts esophagus (BE) with specialized intestinal metaplasia (SIM) is at high risk of esophageal adenocarcinoma. Magnified endoscopy with narrow band imaging (ME‐NBI) can be useful for detecting this condition. In addition to pit patterns, light blue crests (LBC), blue‐whitish patchy areas on the metaplastic epithelia of the stomach, can predict SIM in BE under ME‐NBI observation.

Characterization of dsRNA-induced pancreatitis model reveals the regulatory role of IFN regulatory factor 2 (Irf2) in trypsinogen5 gene transcription

Mice deficient for interferon regulatory factor (Irf)2 (Irf2−/− mice) exhibit immunological abnormalities and cannot survive lymphocytic choriomeningitis virus infection. The pancreas of these animals is highly inflamed, a phenotype replicated by treatment with poly(I:C), a synthetic double-stranded RNA. Trypsinogen5 mRNA was constitutively up-regulated about 1,000-fold in Irf2−/− mice compared with controls as assessed by quantitative RT-PCR. Further knockout of IFNα/β receptor 1(Ifnar1) abolished poly(I:C)-induced pancreatitis but had no effect on the constitutive up-regulation of trypsinogen5 gene, indicating crucial type I IFN signaling to elicit the inflammation. Analysis of Ifnar1−/− mice confirmed type I IFN-dependent transcriptional activation of dsRNA-sensing pattern recognition receptor genes MDA5, RIG-I, and TLR3, which induced poly(I:C)-dependent cell death in acinar cells in the absence of IRF2. We speculate that Trypsin5, the trypsinogen5 gene product, leaking from dead acinar cells triggers a chain reaction leading to lethal pancreatitis in Irf2−/− mice because it is resistant to a major endogenous trypsin inhibitor, Spink3.

Magnifying videoendoscopic findings of Peyer’s patches in the terminal ileum of Crohn’s disease

It has been reported that Crohn’s disease initially occurs as tiny aphthoid lesions at the sites of lymphoid follicles in the gastrointestinal tract.1–3 The follicle-associated epithelium (FAE) of the gut-associated lymphoid tissues such as Peyer’s patches (PPs)3,4 is a single layer of epithelial cells covering each follicle and forms a dome between the surrounding villi.3,4 Endoscopic observation of PPs in patients with Crohn’s disease has rarely been performed in clinical settings.1–3,5,6 A total of seven patients with active Crohn’s disease and 19 age- and sex-matched healthy controls were enrolled. Chromoendoscopy was carried out with crystal violet and/or indigo carmine to identify PPs. The FAE on the domes of PPs was examined by magnifying endoscopy. The macroscopic appearance of PPs was classified into two categories, a nodular or convolute elevation pattern (E type, fig …

Ventilation-Based Decellularization System of the Lung

Abstract The demand for donated organs greatly exceeds the availability. Alternatives to organ donation, such as laboratory-engineered organs, are therefore being developed. One approach is to decellularize the organ and reseed it with selected cells, ideally from the organ recipient. Organ decellularization has typically been attempted by the administration of detergents into vessels such as the portal vein in the liver. However, in the case of the lung, the airway provides another potential administration route, because it has a wide contact area between cells and detergents in the tracheal tree and alveoli. In the present study, we introduce a novel ventilation-based decellularization system for the lung and compare its efficacy to ordinary decellularization systems administering detergent through the pulmonary artery. Rat lungs were decellularized using 500 mL of 3-[(3-cholamidopropyl) dimethylammonio]-1-Propanesulfonate (CHAPS) decellularization solution administrated through the pulmonary artery (vessel group) or through the trachea (airway group). The vessel group was infused CHAPS solution using a gravitational pressure head of 20 cmH2O. The airway group was infused with the detergent using negative pressure and positive end-expiratory pressure, for a volume 10cc with each inspiration in a bioreactor. Pathological and immunohistochemical findings indicated that components of the extracellular matrix (ECM), including proteoglycans, elastic fibers, fibronectin, and laminin, were more decreased in the airway group than in the vessel group. Western blot analysis showed that MHC class I antigen and β-actin were not detected in both decellularized groups. A collagen assay showed that collagen was 70% preserved in both groups compared to native lung. Glycosaminoglycan (GAG) and DNA assays showed that GAG and DNA contents were strongly diminished in both decellularized groups, but those contents were smaller in the airway group than in the vessel group. Accordingly, the alveolar wall was thinner on electron microscopy, and DNA remnants were not observed in the airway group. Infusion of red blood cells indicated that capillary walls were preserved without blood leakage in both groups. In conclusion, we describe a novel approach for decellularization through the airway that represents a more stringent method for both DNA and ECM removal, with capillary wall preservation.

VISUALIZATION OF ULTRAVIOLET LIGHT‐INDUCED THYMINE DIMERS IN DNA BY IMMUNOELECTRON MICROSCOPY

Abstract— To see the damage of DNA due to ultravoilet‐B more distinctly, immunoelectron microscopic studies using a monoclonal antibody against cyclobutane‐type thymine dimers were performed. As a result, we could detect the existence of thymine dimers on human genomic DNA and pUC18 plasmid DNA visually. This technique can be useful to locate the photoproducts formed on DNA.

Coexpression of Ang1 and Tie2 in Odontoblasts of Mouse Developing and Mature Teeth—A New Insight into Dentinogenesis

Angiopoietin-1 regulates vascular angiogenesis and stabilization, and is reported to promote bone formation by facilitating angiogenesis. To estimate the role of Ang1 in odontogenesis, we explored the distribution of Ang1 and the receptor, Tie2 in the mouse developing and mature first molar of the mandible. At embryonic day 18, when differentiation of odontoblasts begins, immunosignals for Ang1 were intensely detected in the basement membrane and the distal side, which faced the basement membrane of odontoblasts. In situ hybridization revealed that Ang1 was expressed in odontoblasts and ameloblasts facing the basement membrane. Tie2 was localized in the distal side of odontoblasts. After birth, Ang1 was detected in the predentin, whereas both Ang1 and Tie2 were colocalized in odontoblasts and odontoblast processes. These distributions were retained up to 8 weeks. In contrast to odontoblasts, ameloblasts, cementoblasts and osteoblasts expressed Ang1 but did not express Tie2. Colocalization of Ang1 and Tie2 in odontoblasts and selective expression of Tie2 in odontoblasts among cells responsible for calcified tissue formation suggested the involvement of autocrine signals of Ang1-Tie2 in dentinogenesis.

Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within.