Takeshi Ohba

Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.

Comparison of panoramic radiography and Water's projection in the diagnosis of maxillary sinus disease

Pantomograms and Waters views were independently reviewed by two radiologists with regard to detection of maxillary sinus abnormalities. Panoramic radiography was found to be a better radiologic approach than the Waters projection for the detection of cystlike densities in the maxillary sinus. Cloudiness of the maxillary sinus and sclerotic change of adjacent bony structures were better demonstrated on the Waters view. These two techniques supplement each other and both should be used in order to obtain a more accurate diagnosis of maxillary sinus disease.

Extracts of Actinobacillus actinomycetemcomitans Induce Apoptotic Cell Death in Human Osteoblastic MG63 Cells

Whether an extracellular component of periodontal-disease-causing bacteria induces apoptotic cell death in bone-related cells is unknown. To study the effects on osteoblasts of extracts obtained from sonicated Actinobacillus actinomycetemcomitans and Prevotella intermedia, we cultured human osteoblastic cell lines MG63 and Saos-2 cells and mouse osteoblastic cell line MC3T3-E1 cells in the presence of such extracts. The addition of the extracts from Actinobacillus actinomycetemcomitans induced cell death in MG63 cells in a dose- and time-dependent fashion over the concentration range of 0.1 to 10 μg/mL. By contrast, the extracts from Prevotella intermedia did not induce cell death in these cells, even in the presence of 10 μg/mL protein. By using the Hoechst 33342 staining technique, we observed marked nuclear condensation and fragmentation of chromatin in MG63 cells treated with the extracts of Actinobacillus actinomycetemcomitans. DNA ladder formation, a hallmark of apoptosis, also was detected in MG63 cells treated with extracts from Actinobacillus actinomycetemcomitans. In MG63 cells, DNA ladder formation was dose-dependent, with a maximal effect at a concentration of 10 μg/mL, and time-dependent, from 12 to 48 hrs. However, the extracts from Prevotella intermedia did not induce DNA fragmentation in MG63, Saos-2, or MC3T3-E1 cells. The extracts from Actinobacillus actinomycetemcomitans did not induce cell death and DNA fragmentation in Saos-2 and MC3T3-E1 cells. Sonicated extracts of Actinobacillus actinomycetemcomitans that had been treated with heat and trypsin did not induce DNA ladder formation in MG63 cells, suggesting that the apoptosis-inducing factors are proteinaceous. Cycloheximide prevented the Actinobacillus actinomycetemcomitans-induced DNA ladder formation in MG63 cells in a dose-dependent fashion, suggesting that new gene transcription and protein synthesis are regulated for Actinobacillus actinomycetemcomitans-induced apoptosis in MG63 cells. Our results indicate that apoptosis in alveolar bone cells induced by Actinobacillus actinomycetemcomitans plays an important role in periodontal diseases.

Assessment of the relationship between the mandibular cortex on panoramic radiographs and the risk of bone fracture and vascular disease in 80-year-olds

OBJECTIVES The objective of this study was to assess cortical measurements on panoramic radiographs in 80-year-old subjects to predict bone fracture and vascular disease risks. METHODS The cortical width and shape (normal cortex, mildly to moderately eroded cortex, and severely eroded cortex) were evaluated on 659 panoramic radiographs obtained from 262 men and 397 women, all of whom were 80 years old. At baseline, a general medical examination, including heel bone density, was performed in all subjects. Fractures and vascular disease occurring within 5 years after the baseline examination were determined in 191 subjects and in 108 subjects who died within 5 years after the baseline examination. RESULTS There were significant correlations between heel bone density and cortical width (r = 0.435, P < .001) and shape (r = 0.231, P < .001). However, cortical measurements on panoramic radiographs were not significantly associated with the occurrence of fractures and vascular disease within 5 years after the baseline examination. CONCLUSIONS Among the elderly, cortical measurements on panoramic radiographs may be associated with bone mineral density and physical activity, but they are not useful markers for the subsequent occurrence of fractures and vascular disease.

Mandibular metastasis of osteogenic sarcoma: Report of a case

Metastasis of osteogenic sarcoma to the mandible is extremely rare. A case of mandibular metastasis of an osteogenic sarcoma of the left fibula is presented. The patient, a 17-year-old Japanese girl, was suffering from a metastatic gingival tumor, 3 by 5 cm. in size, with ulceration. Eventually, there were multiple metastases not only to the mandible but also to the thoracic vertebrae, lumbar vertebrae, sacrum, zygomatic bone, clavicle, sternum, humerus, rib, femur, tibia, lung, and meninges.

Panoramic roentgen anatomy of the maxillary sinus

A dry maxillozygoma was radiographed with a Panex panoramic unit with lead foil and wires on the anatomic landmarks. The medical border of the maxillary sinus on the pantomogram is seen at the angle of the anterior and medial walls. The lateral border of the maxillary sinus consists of the maximum posterior convexity of the posterior, and medial walls are recognized as anatomic landmarks only when radiopaque materials are attached to individual walls. Most of the anterior and posterior walls of the maxillary sinus are superimposed upon the medial wall in the pantomogram. The anterior wall occupies the lateral third of the maxillary sinus. Anterior, posterior, and medial walls do not appear as anatomic landmarks on the pantomogram.

Upregulation of the expression of Fas antigen and Fas ligand in a human submandibular gland ductal cell line by okadaic acid

Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to the receptor leads to receptor activation and the induction of intracellular signals that result in apoptotic cell death. In the present study, the expression of mRNA and proteins of Fas receptor and Fas ligand were examined in human submandibular gland ductal (HSG) cells treated with okadaic acid by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Six hundred and eighty-two bp of the PCR product of Fas receptor mRNA was detected in HSG cells and a protein with an estimated molecular weight of 58,000 was expressed in HSG cells. Treatment of HSG cells with an agonistic anti-Fas monoclonal antibody resulted in death of HSG cells, indicating that the functional Fas receptor protein is expressed in HSG cells. Fas receptor protein expression stimulated by okadaic acid was elevated in a dose- and time-dependent manner, with maximal expression at 20 nM and 48 h treatment. Fas ligand mRNA was also detected constitutively in HSG cells by RT-PCR. Okadaic acid stimulated the expression of Fas ligand protein in HSG cells in a time-dependent manner, while the expression of the ligand was low in untreated HSG cells. The molecular weight of Fas ligand was estimated as 68,000. An antagonistic anti-Fas ligand monoclonal antibody prevented okadaic acid-induced death in HSG cells in a dose-dependent fashion as determined by WST-1 assay. The results indicate that the expression of Fas receptor and ligand is regulated by protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in HSG cells. The results also suggest that the Fas receptor-ligand system might regulate apoptosis in HSG cells.

Postoperative maxillary cyst.

The postoperative maxillary cyst develops in the maxillary sinus between the ages of 30 and 40 in persons who received sinus surgery 10 to 20 years earlier. Etiology of the cystic lesion appears to be derived from the infected sinus mucosa left after a sinus operation. Chief complaints of postoperative maxillary cysts are swelling or pain in the buccal region, discomfort of the maxilla or maxillary teeth and exophthalmos. Characteristic panoramic radiographic findings of the postoperative maxillary cyst are a monolocular cystic radiolucency with a well-defined margin and surrounding sclerotic bone in the floor of the maxillary sinus.

Significance of Frequency-Selective Fat Saturation T2-Weighted MR Images for the Detection of Bone Marrow Edema in the Mandibular Condyle

Abstract The objective of this study was to evaluate the utility of frequency-selective fat saturation (FS) T2-weighted images (T2WI) for the detection of bone marrow edema in the mandibular condyle. MR evidence of bone marrow abnormalities was examined on the set of FS T2WI and conventional T1WI or of conventional T2WI and T1WI in 200 patients with temporomandibular joint (TMJ) related pain. Other parameters studied were TMJ effusion, disk displacement categories, and cortical bone abnormalities. The detection rate and area of bone marrow edema by FS T2WI and T1WI were significantly greater than those assessed by conventional T2WI and T1WI. The correlation between bone marrow abnormalities on FS T2WI and T1WI and pain was significantly stronger than with conventional T2WI and T1WI. This study confirms that FS T2WI is useful for the detection of the “edema pattern” in the mandibular condylar associated with TMJ-related pain.

Inhibitors of protein synthesis and RNA synthesis protect against okadaic acid-induced apoptosis in human osteosarcoma cell line MG63 cells but not in Saos-2 cells

Abstract: In a previous study, we demonstrated that the protein phosphatase inhibitors, okadaic acid and calyculin A, induced apoptosis in human osteosarcoma cell lines, Saos-2 and MG63 cells. In the present study, to determine if new gene transcription and protein synthesis are required for okadaic acid-induced apoptosis in Saos-2 and MG63 cells, the cells were treated for 48 h with varying concentrations of the inhibitors of protein or RNA synthesis, i.e., cycloheximide, actinomycin D, and puromycin, in the presence of a fixed dose of okadaic acid. All these reagents in different concentrations prevented the okadaic acid-induced apoptosis in MG63 cells in a dose-dependent fashion. The same concentrations of cycloheximide, actinomycin D, or puromycin alone did not induce any apoptotic features in MG63 cells. However, not all the aforementioned reagents affected okadaic acid-induced apoptosis in Saos-2 cells. Okadaic acid-induced and cycloheximide-prevented apoptosis was shown by phase-contrast microscopy, WST-1 assay, direct visualization of nuclear condensation and fragmentation of chromatin, and the characteristic DNA ladder formation on agarose gel electrophoresis. The present results indicate that the induction of new cell death genes and ongoing protein synthesis may have a role in okadaic acid-induced apoptosis in MG63 cells and that such proteins are not required in Saos-2 cells.