Improvement in the facial profile of an adult patient with bimaxillary protrusion : A case report

症例報

Immortalization of Fetal Bovine Colon Epithelial Cells by Expression of Human Cyclin D1, Mutant Cyclin Dependent Kinase 4, and Telomerase Reverse Transcriptase: An In Vitro Model for Bacterial Infection.

Cattle are the economically important animals in human society. They are essential for the production of livestock products such as milk and meats. The production efficiency of livestock products is negatively impacted by infection with zoonotic pathogens. To prevent and control infectious diseases, it is important to understand the interaction between cattle tissue and pathogenic bacteria. In this study, we established an in vitro infection model of an immortalized bovine colon-derived epithelial cell line by transducing the cells with lentiviral vectors containing genes encoding cell cycle regulators cyclin D1, mutant cyclin dependent kinase 4 (CDK4), and human telomerase reverse transcriptase (TERT). The established cell line showed continuous cell proliferation, expression of epithelial markers, and an intact karyotype, indicating that the cells maintained their original nature as colon-derived epithelium. Furthermore, we exposed the established cell line to two strains of Salmonella enterica and EHEC. Interestingly, S. Typhimurium showed higher affinity for the established cell line and invaded the cytoplasm than S. Enteritidis. Quantitative RT-PCR revealed that gene expression of Toll-like receptor 1 (TLR1), TLR 2 and TLR 3, whereas TLR 4, 5 and 6 were not detectable in established cells. Our established immortalized colon-derived epithelial cell should be a useful tool for studies evaluating the molecular mechanisms underlying bacterial infection

Electrophysiological and Morphological Properties of α and γ Motoneurons in the Rat Trigeminal Motor Nucleus

The muscle contraction during voluntary movement is regulated by activities of α- and γ-motoneurons (αMNs and γMNs, respectively). The tension of jaw-closing muscles can be finely tuned over a wide range. This excellent function is likely to be achieved by the specific populations of αMNs innervating jaw-closing muscles. Indeed, we have recently demonstrated that in the rat dorsolateral trigeminal motor nucleus (dl-TMN), the size distribution of αMNs was bimodal and the population of smaller αMNs showed a size distribution similar to that of γMNs, by immunohistochemically identifying αMNs and γMNs based on the expressions of estrogen-related receptor gamma (Err3) and neuronal DNA binding protein NeuN together with ChAT. This finding suggests the presence of αMNs as small as γMNs. However, differences in the electrophysiological membrane properties between αMNs and γMNs remain unknown also in the dl-TMN. Therefore, in the present study, we studied the electrophysiological membrane properties of MNs in the dl-TMN of infant rats at postnatal days 7–12 together with their morphological properties using whole-cell current-clamp recordings followed by immunohistochemical staining with an anti-NeuN and anti-ChAT antibodies. We found that the ChAT-positive and NeuN-positive αMNs were divided into two subclasses: the first one had a larger cell body and displayed a 4-aminopyridine (4-AP)-sensitive current while the second one had a smaller cell body and displayed a less prominent 4-AP-sensitive current and a low-threshold spike, suitable for their orderly recruitment. We finally found that γMNs showing ChAT-positive and NeuN-negative immunoreactivities had smaller cell bodies and displayed an afterdepolarization mediated by flufenamate-sensitive cation current. It is suggested that these electrophysiological and morphological features of MNs in the dl-TMN are well correlated with the precise control of occlusion

Toll-like receptors (TLRs) gene expression in established BFCE-K4DT cells.

<p>+: positive,</p><p>-: negative</p><p>Toll-like receptors (TLRs) gene expression in established BFCE-K4DT cells.</p

Fluorescence microscopic images of cells subjected to the adhesion and invasion assay.

<p><i>S</i>. Typhimurium (a-d: low-power fields and e-h; high-power fields), <i>S</i>. Enteritidis (i-l) after infection, and non-infected control (m-p)) in BFCE-K4DT cells (passage number = 15). Total bacteria were stained green (a, e, i, and m; black arrows), adherent bacteria were stained red (b, f, j, n; white arrows), nuclei of cells were stained blue, and merge images of extracellular bacteria (stars) and intracellular bacteria (black arrows) appear yellow and green respectively (d, h, l, p). Each staining was carried for 3 times and representative pictures were shown here.</p

Results of the immunoblotting analysis and TRAP assay.

<p>(A) Detection of CDK4 and cyclin D1 by immunoblotting. Lane 1, BFCE primary cells; Lanes 2 and 3, BFCE-K4DT cells. (B) Detection of telomerase activity by TRAP assay. Lane 1, 1 kb ladder marker; lane 2, CHAPS buffer as a negative control; lane 3, 293T cells as a positive control; lane 4, BFCE primary cells; lane 5 and 6, BFCE-K4DT cells. The 6-bp ladders were detectable in lanes of positive control and BFCE-K4DT cells. Each experiment was performed in duplicates after cloning (primary cells: 2 times passage, K4DT cells: 15 times passage) and representative data were shown here.</p

Quantification of bacterial adhesion and invasion and SEM microscopy.

<p>(A) Total <i>S</i>. Enteritidis or <i>S</i>. Typhimurium were harvested after each infection time (30, 60, 120 min) and plated in agar dishes to count the colony number (gray bars). Invasive bacteria were also harvested after 2 h gentamycin treatment at the each time point and count the colony by same procedures as total bacteria (black bars). (B) EHEC adhesion to BFCE-Primary cells (gray bars) and BFCE-K4DT cells (black bars) were also quantification by same methods as <i>Salmonella</i> infection. (C) SEM observations revealed the adhesion of <i>S</i>. Enteritidis (a), <i>S</i>. Typhimurium (b) and EHEC (c) to established BECE-K4DT cells (bars: 10 μm). Experiments were carried out in triplicate, and the averages and standard deviations (SD) were calculated (A and B) and representative figures were shown (C).</p

Karyotype analysis of established BFCE-K4DT cells.

<p>All mitotic chromosome spreads (50 of 50 examined) from BFCE-K4DT cells showed a 2n = 60XY pattern. This analysis was performed by using cloned cells after 15 times passage.</p

Fluorescent immunohistochemical staining for detection of epithelial markers.

<p>BFCE-K4DT cells (passage number = 15) and BFCE-Primary cells (passage number = 2) were positive for E-cadherin and Cytokeratin-8, whereas BFF NCC cells (passage number was unknown) were negative for both markers. Scale bars: 50 μm. Each staining was carried for 3 times and representative pictures were shown here.</p