Immunohistochemical study of calpain-mediated alpha-crystallin proteolysis in the UPL rat hereditary cataract

The purpose of this study is to examine if microencapsulated PC12 cells may provide long term effects to the hemiparkinsonian rats. A modified technique was used to encapsulate PC12 cells into gelled microspheres. We found that the PC12 cells can survive in the modified microcapsules in vitro. Most of the PC12 cells formed cluster 3 weeks after incubation. The PC12 cell-loaded microcapsules were also examined in vitro. Adult Sprague-Dawley rats, anesthetized with chloral hydrate, were injected unilaterally with 6-hydroxydopamine into the medial forebrain bundle. The effectiveness of this lesion was tested by measuring apomorphine or methamphetamine-induced rotation one month after lesioning. The unilaterally lesioned rats were transplanted with microencapsulated PC12 cells. Results showed that apomorphine and methamphetamine-induced rotations were greatly suppressed after transplantation. One year after the grafting, the animals were anesthetized with urethane for the voltammetric study. Low dose of KCl was directly injected into the grafted striatum through pressure microejection. We found that KCl-induced DA release, as measured by voltammetric techniques, was regenerated in the striatum. The animals were later sacrificed for histological examination. We found that capsules were present in the lesioned striatum one year after grafting. Most of the capsules contained no PC12 cell. However, some capsules were filled entirely with PC12 cells. Taken together, our data suggested that PC12 cells can survive in the capsule in vitro and may provide long-term dopaminergic effects to the hemiparkinsonian rats.     

ONO-5046 is a potent inhibitor of neutrophil elastase in human pleural effusion after lobectomy

The imbalance of neutrophil elastase and alpha1-antitrypsin in pleural effusion after lobectomy and the effects of the neutrophil elastase inhibitors, sodium N-[2-[4-(2,2-Dimethylpropionyloxy)phenyl-sulfonylamino]benzo yl]aminoacetic acid (ONO-5046) and purified alpha1-antitrypsin, on neutrophil elastase activity were determined. The amount of neutrophil elastase complexed to alpha1-antitrypsin, measured by an enzyme-linked immunosorbent assay, was 170 times higher in pleural effusion than in blood 3 h after lobectomy. The alpha1-antitrypsin levels measured by laser nephelometry did not increase in either blood or pleural effusion. Although neutrophil elastase activity, measured by the hydrolysis of succinyl-(Ala)3-p-nitroanilide, was not detected in blood, it was increased in pleural effusion 3 h and 24 h after lobectomy. ONO-5046, but not alpha1-antitrypsin, reduced the neutrophil elastase activity in pleural effusion. There is an imbalance of neutrophil elastase and alpha1-antitrypsin in pleural effusion after lobectomy. ONO-5046 is a potent inhibitor of neutrophil elastase activity in human pleural effusion.     

Quantitative in situ hybridization of three gonadotropin-releasing hormone-encoding mRNAs in castrated and progesterone-treated male tilapia

We investigated the effects of castration and progesterone administration on the three gonadotropin-releasing hormone (GnRH)-encoding mRNAs in sexually mature male tilapia Oreochromis niloticus. In situ hybridization histochemistry was performed using 35S-labeled antisense oligonucleotide probes complementary to salmon-, seabream-, and chicken II-GnRH cDNAs to quantify cellular GnRH mRNA expression in the terminal nerve ganglia (nucleus olfactoretinalis), preoptic area, and midbrain tegmentum of animals castrated for 2 weeks and injected intraperitoneally with sesame oil or progesterone. Castration significantly elevated salmon-GnRH mRNA but not seabream- or chicken II-GnRH mRNA levels. Progesterone treatment had no effect on salmon-, seabream-, or chicken II-GnRH mRNA levels. Comparisons between intact, castrated, and progesterone-treated animals showed no change in the total volume of nucleus olfactoretinalis, cell sizes, and total numbers of cells expressing GnRH mRNA within the midbrain and preoptic area. These results demonstrate that salmon-GnRH but not seabream- or chicken II-GnRH-synthesizing neurons are under a gonadal steroid negative feedback control and that progesterone might not be the main hormone regulating the three GnRH-encoding mRNAs in the male tilapia.     

Cathodoluminescence study of AlGaAs/GaAs multilayers grown on ridge-type triangles on GaAs (111)A substrates

We have studied the cathodoluminescence of AlxGa1-xAs/GaAs multilayers grown on ridge-type triangles by molecular beam epitaxy. The compositional variation of Al, as well as the distribution of impurity and/or defect, was revealed by variations in the cathodoluminescence spectra and images. The Al composition in an AlxGa1-xAs layer was highest in the (111)A facet and decreased in the order (100), (411)A, (111)-delta and (110) facets. On the other hand, the carbon concentration was highest in the (411)A facet and decreased in the order (111)A, (111)-delta, (100) and (110) facets. It should be noted that the (111)-delta facet has a significant effect on the redistribution of Al. Although our ridge-type triangles are rather large for the quantum structures, these data have elucidated the self-organization mechanism of the AlxGa1-xAs/GaAs system and have yielded information on the design of quantum structures. We conclude that cathodoluminescence observation is a powerful tool for studying the compositional variation or band structure of three-dimensional microscale or nanoscale construction.     

Mössbauer studies and magnetic properties of SnO2 doped with Fe

Transparent conducting SnO₂ powders doped with 10% Fe content were prepared by a polymerized complex method under acidic solutions, and annealed finally at 550 °C, and at 600 °C. These samples were characterized by X-ray diffraction, magnetization, and Mössbauer spectrometry at room temperature. Rutile SnO₂ phase was obtained for both samples, and the crystallite sizes were in the range of 13-14 nm. Both samples exhibit magnetization and the saturation magnetization was smaller for the sample annealed at 600 °C than for sample annealed at 550 °C. Room temperature Mössbauer spectra for both samples showed the presence of two different paramagnetic iron sites but no magnetic sextets. These results suggest that ferromagnetism originates from magnetic defects and not directly from iron ions.     

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.     

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.