Identification of SYS1 as a Host Factor Required for Shiga Toxin-Mediated Cytotoxicity in Vero Cells

Shiga toxin (STx) or Vero toxin is a virulence factor produced by enterohemorrhagic Escherichia coli. The toxin binds to the glycosphingolipid globotriaosylceramide (Gb3) for its entry, and causes cell death by inhibiting ribosome function. Previously, we performed a loss-of-function screen in HeLa cells using a human CRISPR knockout (KO) library and identified various host genes required for STx-induced cell death. To determine whether this library targeted to the human genome is applicable to non-human primate cells and to identify previously unrecognized factors crucial for STx-induced cell death, we herein performed a similar screen in the African green monkey kidney-derived Vero C1008 subline. Many genes relevant to metabolic enzymes and membrane trafficking were enriched, although the number of enriched genes was less than that obtained in the screening for HeLa cells. Of note, several genes that had not been enriched in the previous screening were enriched: one of these genes was SYS1, which encodes a multi-spanning membrane protein in the Golgi apparatus. In SYS1 KO Vero cells, expression of Gb3 and sphingomyelin was decreased, while that of glucosylceramide and lactosylceramide was increased. In addition, loss of SYS1 inhibited the biosynthesis of protein glycans, deformed the Golgi apparatus, and perturbed the localization of trans-Golgi network protein (TGN) 46. These results indicate that the human CRISPR KO library is applicable to Vero cell lines, and SYS1 has a widespread effect on glycan biosynthesis via regulation of intra-Golgi and endosome–TGN retrograde transports.     

Involvement of the adrenal glands and testis in gap junction formation via testosterone within the male rat anterior pituitary gland

We investigated the influence of testicular and adrenal androgens on the presence of gap junctions between folliculo‐stellate cells in the anterior pituitary glands of 60‐day‐old Wistar‐Imamichi strain male rats. The animals were separated into six groups: Group A served as the controls and had free access to a normal diet and water, Group B was given a normal diet and 0.9% NaCl for their drinking water as the controls of adrenalectomized groups, Group C was castrated, Group D was adrenalectomized, Group E was both castrated and adrenalectomized, and Group F was also both castrated and adrenalectomized. In addition, the animals of Group F were administered a dose of testosterone that is known to produce high physiological levels of the hormones in plasma. Five rats from each group were sacrificed 1, 2, 3, 4, 5, 6, and 7 days after their respective operation, and the anterior pituitary glands were removed and prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo‐stellate cells per intersected follicle profile. Simultaneous removal of adrenal glands with castration resulted in a significantly decrease in the number of gap junctions, whereas the administration of testosterone to these rats compensated for this change. These observations indicate that the preservation of gap junctions between folliculo‐stellate cells is mainly dependent on androgens from both the testes and adrenal glands in adult male rats. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Effect of Cation Doping on the Superplastic Flow in Yttria-Stabilized Tetragonal Zirconia Polycrystals

The superplastic characteristics of various cation-doped yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) were examined. For 1 mol% cation doping the true stress of Y-TZP is very dependent on the ionic radii of the doped cations; for instance, smaller cation radii give rise to lower true stress when compared with the other compositions for the same grain size, strain rate, and testing temperature. The altered true stress level must be due to the change in diffusivity of the accommodation process for grain boundary sliding caused by the addition of cations in ZrO₂. The strain to failure of the doped zirconia is affected by both ionic radius and valence of the dopant cations.     

Low-temperature thermionic emission from nitrogen-doped nanocrystalline diamond films on n-type Si grown by MPCVD

Temperature-dependent emission current–voltage measurements were carried out for nitrogen (N)-doped nanocrystalline diamond (NCD) films grown on n-type Si substrates by microwave plasma-assisted chemical vapor deposition (MP-CVD). Low threshold temperature (~ 260 °C) and low threshold electric field (~ 5 × 10^− 5 V/µm) were observed. Both the temperature dependence and the electric field dependence have shown that the obtained emission current was based on electron thermionic emission from N-doped NCD films. We have also studied the relation between nitrogen concentration and the saturation emission current. The saturation current obtained was as high as 1.4 mA at 5.6 × 10^− 3 V/µm at 670 °C when the nitrogen concentration was 2.4 × 10²⁰ cm^− 3. Low value of effective work function (1.99 eV) and relatively high value of Richardson constant (~ 70) were estimated by well fitting to Richardson–Dushman equation. The results of smaller φ and larger A′ suggest that N-doped NCD has great possibility of being a highly efficient thermionic emitter material.     

Use of cysteine to remove mercury from shark muscle

The efficiency of cysteine to remove mercury from sliced and minced shark was evaluated. Treatment with 0.5% cysteine at pH 2.0–2.5 and subsequent washing with sodium chloride (5%) failed to remove the mercury from the sliced shark. The efficiency of 0.5% cysteine at pH 7.0 in removing mercury from minced shark was 40–45% (dry material), obtained by two of the three methods studied. This removal rate was considered relatively low for it failed to attend to the practical proposal of decontaminating species of fish highly contaminated by mercury. The need for fuller understanding of the mechanisms involved in the removal of mercury by cysteine was also considered.     

Solute segregation at grain boundaries in superplastic SiO2-doped TZP

Grain boundary structure, chemical composition, and bonding state in superplastic SiO₂-doped TZP and undoped TZP were investigated by high resolution electron microscopy (HREM), energy dispersive X-ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS) using a field emission type transmission electron microscope (FE-TEM). No amorphous phase was observed at any grain boundaries in either SiO₂-doped TZP or undoped TZP. Yttrium ions segregated over a width of 4–6 nm at grain boundaries in both materials, and silicon ions segregated over a width of 5–8 nm at grain boundaries in SiO₂-doped TZP. The average dihedral angle between grain boundaries in SiO₂-doped TZP was as high as 80°, which agreed well with the fact that no grain boundary had glass phase. The strain energy is accumulated by the dissolution of silicon ions into the tetragonal zirconia lattice. However, the grain boundary energy of SiO₂-doped TZP is likely to be low enough to compensate the increase of strain energy near grain boundaries. OK-edge EELS spectra taken from grain boundaries in SiO₂-doped TZP were shifted 3–4 eV to the higher energy side in comparison with those from the grain interior. This may suggest that the chemical bonding is strengthened at grain boundaries by the presence of solute silicon. The strengthening may be responsible for the enhanced superplasticity in SiO₂-doped TZP.     

Phenotypic changes in lipopolysaccharide-treated cloned cells derived from transplantable rat malignant fibrous histiocytoma

To investigate a possible phenotypic modulation, MT-8L and MT-9L cells were induced by in vitro culture of undifferentiated MT-8 and fibrohistiocytic MT-9 cells, which had been established from a rat malignant fibrous histiocytoma (MFH), in the medium containing 10 micrograms lipopolysaccharide (LPS)/ml. MT-8L and MT-9L gave greater positive reactions for histiocytic lysosomal markers and showed ultrastructures of histiocytic natures. In MT-8L, alpha-smooth muscle actin-positive myofibroblastic cells also significantly increased in number. MT-8L expressed both histiocytic and myofibroblastic phenotypes. MT-8L-induced tumors consisted mainly of storiform type MFH, differing from undifferentiated sarcoma type induced by MT-8. MT-9L and MT-9 tumors showed a storiform pattern. A phenotypic modulation of MFH cells was easily induced by LPS treatment.