Enterocin X,a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides

Enterocin X, composed of two antibacterial peptides (Xα and Xβ), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xα and Xβ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.Bacteriocins are gene-encoded antibacterial peptides and proteins. Because of their natural ability to preserve food, they are of particular interest to researchers in the food industry. Bacteriocins are grouped into three main classes according to their physical properties and compositions (11, 12). Of these, class IIb bacteriocins are thermostable non-lanthionine-containing two-peptide bacteriocins whose full antibacterial activity requires the interaction of two complementary peptides (8, 19). Therefore, two-peptide bacteriocins are considered to function together as one antibacterial entity (14).Enterocins A and B, first discovered and identified about 12 years ago (2, 3), are frequently present in Enterococcus faecium strains from various sources (3, 5, 6, 9, 13, 16). So far, no other bacteriocins have been identified in these strains, except the enterocin P-like bacteriocin from E. faecium JCM 5804^T (18). Here, we describe the characterization and genetic identification of enterocin X in E. faecium KU-B5. Enterocin X (identified after the enterocin P-like bacteriocin was discovered) is a newly found class IIb bacteriocin in E. faecium strains that produce enterocins A and B.     

TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation.Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown.Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGFβ enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells.Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGFβ might be an important pathway of gastric cancer cell proliferation by TGFβ.     

Neurodegeneration in mnd2 mutant mice is not prevented by parkin transgene

Parkinson’s disease (PD) is a common neurodegenerative disorder. The motor neuron degeneration 2 mutant (mnd2) mouse is considered to be an animal model of PD, and exhibits striatal neuron loss, severe muscle wasting, weight loss and death before 40 days of age. We found for the first time that parkin expression was decreased in the mnd2 mouse brain. Since parkin is a crucial protein for PD, the neurodegenerative disorder in mnd2 mice may be caused by parkin protein loss. We therefore examined whether compensation of parkin protein prevents neurodegenerative disorders in mnd2 mice by generating parkin-transgenic (parkin-Tg) mnd2 mice. However, both parkin-Tg mnd2 mice and mnd2 mice were smaller than wild type mice. In muscle strength and survival rate, parkin-Tg mnd2 mice showed similar values to mnd2 mice. Our data suggest that repression of parkin protein does not play a major role in neurodegeneration of mnd2 mice and administration of parkin protein does not rescue mnd2 mice.     

Bifurcate effects of glucose on caspase-independent cell death during hypoxia

We investigated the effect of glucose on hypoxic death of rat cardiomyocyte-derived H9c2 cells and found that there is an optimal glucose concentration for protection against hypoxic cell death. Hypoxic cell death in the absence of glucose is accompanied by rapid ATP depletion, release of apoptosis-inducing factor from mitochondria, and nuclear chromatin condensation, all of which are inhibited by glucose in a dose-dependent manner. In contrast, excessive glucose also induces hypoxic cell death that is not accompanied by these events, suggesting a change in the mode of cell death between hypoxic cells with and without glucose supplementation.     

Highly efficient rescue of dengue virus using a co-culture system with mosquito/mammalian cells

The production rate of dengue viruses (DENVs), especially low-passage virus isolates, is low, and, therefore, the isolates are generally used only after several passages. However, in vitro passages could induce mutation(s). In this study, we established a system for the characterization of low-passage viral isolates using an infectious cDNA clone. We used R05-624, a plaque derived from type 2 (DENV-2) Thai strain, for the construction of the cDNA clone, named pmMW/R05-624. We found that transfection of both of mammalian Vero cells and mosquito C6/36 cells with viral RNA derived from the cDNA clone produced a significant amount of progeny virus: 3.2 × 10⁶ focus-forming units (FFU) production per ml of cultured fluid only 3 days after transfection with 2 μg RNA. Conversely, no detectable level of viruses was produced by conventional methods using a single cell line, Vero or C6/36. When this system was applied for the characterization of eight low-passage clinical viral isolates by placing their 5′-half or 3′-half in the above cDNA clone, we found that all the isolates, except for L04-225, produced similar levels of progeny virus. Among a total of eight cDNA clones reconstructed with the NS4A-3′NCR region derived from L04-225, one clone carried an insertion and produced a low level of progeny virus. Thus, our system to efficiently rescue clinical samples or low-passage viral isolates could be useful for assessing the virological and molecular characteristics of DENV that could be related to disease pathogenesis.     

Generation of functional gut-like organ from mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have the pluripotency to differentiate into broad spectrum derivatives of all three embryonic germ layers. However, the in vitro organ differentiation potential of iPS cells to organize a complex and functional “organ” has not yet been demonstrated. Here, we demonstrate that mouse iPS cells have the ability to organize a gut-like organ with motor function in vitro by a hanging drop culture system. This “induced gut (iGut)” exhibited spontaneous contraction and highly coordinated peristalsis accompanied by a transportation of contents. Ultrastructural analysis identified that the iGut had large lumens surrounded by three distinct layers (epithelium, connective tissue and musculature). Immunoreactivity for c-Kit, a marker of interstitial cells of Cajal (ICCs, enteric pacemaker cells), was observed in the wall of the lumen and formed a distinct and dense network. The neurofilament immunoreactivity was identified to form large ganglion-like structures and dense neuronal networks. The iGut was composed of all the enteric components of three germ layers: epithelial cells (endoderm), smooth muscle cells (mesoderm), ICCs (mesoderm), and enteric neurons (ectoderm). This is the first report to demonstrate the in vitro differentiation potential of iPS cells into particular types of functional “organs.” This work not only contributes to understanding the mechanisms of incurable gut disease through disease-specific iPS cells, but also facilitates the clinical application of patient-specific iPS cells for novel therapeutic strategies such as patient-specific “organ” regenerative medicine in the future.     

Critical function of death-associated protein 3 in T cell receptor-mediated apoptosis induction

Death-associated protein 3 (DAP3) is crucial for promoting apoptosis induced by various stimulations. This report demonstrates that DAP3 is also important for T cell receptor (TCR)-mediated apoptosis induction in immature thymocytes. Enforced expression of DAP3 accelerated the negative selection in developing thymocytes, using the reaggregate thymus organ culture system. In addition, expression of DAP3 accelerated TCR-mediated apoptosis induction in DO11.10 cells. We also demonstrated that DAP3 translocates into the nucleus during TCR-mediated apoptosis in a Nur77 dependent manner. It is concluded that DAP3 is critical for TCR-mediated induction of apoptosis at the downstream of Nur77.     

Estimating stand volume in broad-leaved forest using discrete-return LiDAR: plot-based approach

Quantitative assessment of forests is important at a variety of scales for forest planning and management. This study investigated the use of small-footprint discrete-return lidar for estimating stand volume in broad-leaved forest at plot level. Field measurements were conducted at 20 sample plots in the study area in western Japan, composed of temperate broad-leaved trees. Five height variables and two density variables were derived from the lidar data: 25th, 50th, 75th, and 100th percentiles, and mean of laser canopy heights as height variables (h ₂₅, h ₅₀, h ₇₅, h ₁₀₀, h _mean); and ground fraction and only-and-vegetation fraction (d _GF, d _OVF) as density variables, defined respectively as the proportion of laser returns that reached the ground, and the proportion of only echoes (i.e., single pulse returns for which the first and last pulses returned from the same point) within vegetation points. In addition, the normalized difference vegetation index (NDVI), which is often used as an estimator for leaf area index (LAI) and above-ground biomass, was derived from multispectral digital imagery as an alternative density variable (d _NDVI). Nonlinear least-square regression with cross-validation analysis was performed with single variables and combinations; a total of 23 models were studied. The best prediction was found when h ₇₅ and d _OVF were used as independent variables, resulting in adjusted R ² of 0.755 and root-mean-square error (RMSE) of 41.90 m³ ha⁻¹, corresponding to 16.4% of the mean stand volume, better than or comparable to the prediction models of previous studies.     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.