Cleavage of Host Cytokeratin-6 by Lysine-Specific Gingipain Induces Gingival Inflammation in Periodontitis Patients

Background/PurposeLysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion.MethodsK6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay.ResultsWe identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359–378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt.ConclusionKgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.     

An Efficient reproductive method for Irs2-/- mice with C57BL/6JJcl genetic background

Efficient reproduction using natural mating and reproduction technology [in vitro fertilization (IVF) and embryo transfer (ET)] was investigated in IRS2 deficient mice with C57BL/6JJcl genetic background (Irs2(-/-) mice) as a typical type 2 diabetes model. From the results using various combinations of Irs2(-/-) and Irs2(-/+) mice, the combination of female Irs2(-/+) x male Irs2(-/-) was found to be more efficient than other combinations. In applications of reproduction technology using IVF and ET, the combination of female Irs2(-/+) x male Irs2(-/-) involves the possibility of Irs2(-/-) production by repeats using female Irs2(-/+) mice. However, reproductive continuity using this combination is difficult because of dependence on human technique and the cost of ET. Therefore, we concluded that Irs2(-/-) mice should be produced by embryo transfer using Irs2(-/-) mice from a colony consisting of female Irs2(-/+) x male Irs2(-/-).     

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.     

Quantitative Analysis of Torso FDG-PET Scans by Using Anatomical Standardization of Normal Cases from Thorough Physical Examinations

Understanding of standardized uptake value (SUV) of 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG-PET) depends on the background accumulations of glucose because the SUV often varies the status of patients. The purpose of this study was to develop a new method for quantitative analysis of SUV of FDG-PET scan images. The method included an anatomical standardization and a statistical comparison with normal cases by using Z-score that are often used in SPM or 3D-SSP approach for brain function analysis. Our scheme consisted of two approaches, which included the construction of a normal model and the determination of the SUV scores as Z-score index for measuring the abnormality of an FDG-PET scan image. To construct the normal torso model, all of the normal images were registered into one shape, which indicated the normal range of SUV at all voxels. The image deformation process consisted of a whole body rigid registration of shoulder to bladder region and liver registration and a non-linear registration of body surface by using the thin-plate spline technique. In order to validate usefulness of our method, we segment suspicious regions on FDG-PET images manually, and obtained the Z-scores of the regions based on the corresponding voxels that stores the mean and the standard deviations from the normal model. We collected 243 (143 males and 100 females) normal cases to construct the normal model. We also extracted 432 abnormal spots from 63 abnormal cases (73 cancer lesions) to validate the Z-scores. The Z-scores of 417 out of 432 abnormal spots were higher than 2.0, which statistically indicated the severity of the spots. In conclusions, the Z-scores obtained by our computerized scheme with anatomical standardization of torso region would be useful for visualization and detection of subtle lesions on FDG-PET scan images even when the SUV may not clearly show an abnormality.     

EML4 promotes the loading of NUDC to the spindle for mitotic progression

Echinoderm microtubule-associated protein (EMAP)-like (EML) family proteins are microtubule-associated proteins that have a conserved hydrophobic EMAP-like protein (HELP) domain and multiple WD40 domains. In this study, we examined the role of EML4, which is a member of the EML family, in cell division. Time-lapse microscopy analysis demonstrated that EML4 depletion induced chromosome misalignment during metaphase and delayed anaphase initiation. Further analysis by immunofluorescence showed that EML4 was required for the organization of the mitotic spindle and for the proper attachment of kinetochores to microtubules. We searched for EML4-associating proteins by mass spectrometry analysis and found that the nuclear distribution gene C (NUDC) protein, which is a critical factor for the progression of mitosis, was associated with EML4. This interaction was mediated by the WD40 repeat of EML4 and by the C-terminus of NUDC. In the absence of EML4, NUDC was no longer able to localize to the mitotic spindle, whereas NUDC was dispensable for EML4 localization. Our results show that EML4 is critical for the loading of NUDC onto the mitotic spindle for mitotic progression.