Contribution to oral and maxillary surgery and surgical technique of osteo-odontokeratoprosthesis in Japan.

Osteo-odontokeratoprosthesis (OOKP) is a technique invented by Strampelli in 1963, in which the patient's own tooth root is used to support an optical cylinder. It uses an autologous tooth-bone-periodontal complex to mount an optical cylinder, which is stabilised by overlying autologous buccal mucosa. OOKP involves two, staged procedures done by ophthalmologists and oral surgeons, and the main contribution from the oral surgeon is during the first stage. To date we have done nine first-stage, and completed eight second-stage, OOKP operations in Japan with a mean follow-up of eight years and 11 months by modifying the original method of the oral surgery. All OOKP procedures were unilateral, and canines were selected as the donor teeth. Patients developed ocular blindness as a result of Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and chemical and thermal burns to the cornea and ocular surface. All eight patients who completed the second stage have been stable, and there have been no major perioperative or postoperative oral complications. The patients' visual acuities were stable with no serious complications. Here we report the technical details of the oral contribution to OOKP.

Omental metastases in clinical stage I endometrioid adenocarcinoma.

The clinical benefit of an omentectomy in endometrioid adenocarcinoma is unclear. The objective of this study was to clarify the significance of an omentectomy performed for clinical stage I endometrioid adenocarcinoma. A prospective study was performed on 134 patients with clinical stage I endometrioid adenocarcinoma who underwent omentectomy in addition to a staging laparotomy between 1998 and 2004: simple total hysterectomy, bilateral salpingo-oophorectomy, pelvic and para-aortic lymph node dissection, and peritoneal cytology. The frequency and prognosis of omental metastases and their relationships with extrauterine spread to other sites were investigated. Omental metastasis was noted in four patients (3.0%). As for extrauterine spread, the positivity rate of lymph node metastases was 13/128 (10.2%), peritoneal cytology was 13/133 (9.8%), and adnexal metastases was 10/134 (7.5%). Omental metastases correlated with peritoneal cytology and adnexal metastases (P < 0.05 for both); however, two of the omental metastases-positive patients were peritoneal cytology negative. All omental metastases-positive patients died shortly after surgery, showing that their prognosis was poor. The omental metastases rate for clinical stage I endometrioid adenocarcinoma was lower than the positive rates for extrauterine spread to other sites; thus, the routine application of omentectomy as a part of a staging laparotomy may not be efficacious. However, omental metastases are a significant poor prognostic factor, and intraoperative examination of the omentum by close inspection and palpation as well as pathologic examination, if possible, may be indicated.

Sodium orthovanadate suppresses DNA damage-induced caspase activation and apoptosis by inactivating p53.

We previously reported that p42/SETbeta is a substrate for caspase-7 in irradiated MOLT-4 cells, and that treating the cells with sodium orthovanadate (vanadate) inhibits p42/SETbeta's caspase-mediated cleavage. Here, we initially found that the inhibitory effect of vanadate was due to the suppression of caspase activation but not of caspase activity. Further investigations revealed that vanadate suppressed upstream of apoptotic events, such as the loss of mitochondrial membrane potential, the conformational change of Bax, and p53 transactivation, although the accumulation, total phosphorylation, and phosphorylation of six individual sites of p53 were not affected. Importantly, vanadate suppressed p53-dependent apoptosis, but not p53-independent apoptosis. Finally, gel-shift and chromatin immunoprecipitation assays conclusively demonstrated that vanadate inhibits the DNA-binding activity of p53. Vanadate is conventionally used as an inhibitor of protein tyrosine phosphatases (PTPs); however, we recommend that the influence of vanadate not only on PTPs but also on p53 be considered before using it.

Inhibition of trigeminal respiratory activity by suckling.

UNLABELLED: The trigeminal motor system is involved in many rhythmic oral-motor behaviors, such as suckling, mastication, swallowing, and breathing. Despite the obvious importance of functional coordination among these rhythmic activities, the system is not well-understood. In the present study, we examined the hypothesis that an interaction between suckling and breathing exists in the brainstem, by studying the respiratory activity in trigeminal motoneurons (TMNs) during fictive suckling using a neonatal rat in vitro brainstem preparation. The results showed that fictive suckling, which was neurochemically induced by bath application of N-methyl-D,L-aspartate and bicuculline-methiodide, or by local micro-injection of the same drugs to the trigeminal motor nucleus, inhibited the inspiratory activities in both respiration TMNs and respiratory rhythm-generating neurons. Under patch-clamp recording, fictive suckling caused membrane potential hyperpolarization of respiration TMNs. We conclude that the brainstem preparation contains an inhibitory circuit for respiratory activity in the trigeminal motor system via the rhythm-generating network for suckling. ABBREVIATIONS: BIC, bicuculline methiodide; GABA, gamma aminobutyric acid; NMA, N-methyl-D,L-aspartate; NMDA, N-methyl-D-aspartate; and TMN, trigeminal motoneuron.

Girdin maintains the stemness of glioblastoma stem cells.

This corrects the article DOI: 10.1038/onc.2011.466.

Regulation of erythrocyte ghost membrane mechanical stability by chlorpromazine.

Chlorpromazine (CPZ), a widely used tranquilizer, is known to induce stomatocytic shape changes in human erythrocytes. However, the effect of CPZ on membrane mechanical properties of erythrocyte membranes has not been documented. In the present study we show that CPZ induces a dose-dependent increase in mechanical stability of erythrocyte ghost membrane. Furthermore, we document that spectrin specifically binds to CPZ intercalated into inside-out vesicles depleted of all peripheral proteins. These findings imply that CPZ-induced mechanical stabilization of the erythrocyte ghost membranes may be mediated by direct binding of spectrin to the bilayer. Membrane active drugs that partition into lipid bilayer can thus induce cytoskeletal protein interactions with the membrane and modulate membrane material properties.

Effect of continuous infusion of propofol on its concentration in blood with and without the liver in pigs.

In living donor liver transplantation, propofol, an intravenous anesthetic drug, has recently been used in both donors and recipients. Propofol is known to have intra- and extrahepatic metabolic pathways, but the effect of its continuous infusion during a long-term anhepatic state is yet to be determined. Recently, we successfully established a simplified pig model of the complete anhepatic state. In this state, we first evaluated hemodynamic parameters relating to the pharmacokinetics of continuously infused propofol (6 mg.kg(-1) x h(-1)). No significant changes in the concentration of hemoglobin or in hemodynamic parameters other than the heart rate were observed during the anhepatic phase when porpofol was continuously infused at the rate that maintains the state. Blood propofol concentrations in the mixed vein, artery, and portal vein were stable during the anhepatic phase. Finally, we confirmed the pharmacokinetics of continuously infused propofol using orthotropic liver transplantation in miniature pigs. The propofol concentration did not change markedly during the transplant procedure. In conclusion, the pharmacokinetics of continuously infused propofol was almost stable with and without the liver in pigs. Extrahepatic metabolism of propofol might help prevent changes in propofol concentrations.

Antibodies raised against peptide fragments of bovine alpha s1-casein cross-react with the intact protein only when the peptides contain both B and T cell determinants.

In order to compare the immune response to peptides with that to the native protein, we used bovine alpha s1-casein (alpha s1-CN), a major milk protein whose conformation seems to be in a highly disordered state in solution, as the model antigen. Firstly, the T and B cell determinants on this protein were localized by a T cell proliferation assay and an enzyme-linked immunosorbent assay (ELISA), using 13 synthetic overlapping peptides encompassing the entire sequence of alpha s1-CN. T cells from alpha s1-CN-primed C3H/He mice, a high responder to alpha s1-CN, strongly proliferated in the presence of peptides 91-110, 61-80 and 46-65. In addition, peptides 151-170, 136-155 and 106-125 were also found to contain T cell determinants. On the other hand, peptides 181-199, 46-65, 76-95 and 106-125 were generally found to be immunodominant B cell determinants, while peptides 121-140, 136-155, 91-110 and 151-170 also had antibody-binding activity. The peptides were then tested for their ability to elicit a specific antibody. This revealed that only peptides 91-110, 106-125, 136-155 and 46-65 were able to produce specific antibodies that bound to the native protein as well as the peptides themselves. These peptides contained both B and T cell determinants on the intact protein. Thus, we confirmed that a peptide corresponding to both T and B cell determinants on alpha s1-CN was capable of eliciting a specific antibody that reacted with the protein as well as the peptide itself.

Cleavage and phosphorylation of XRCC4 protein induced by X-irradiation.

We report the p35 and p60 forms of XRCC4 protein, appearing in human leukemia MOLT-4 or U937 cells following X-irradiation or hyperthermia. p35 appeared in conjunction with the cleavage of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the fragmentation of internucleosomal DNA, and was suppressed by Ac-DEVD-CHO. p35 was also produced in vitro by treating MOLT-4 cell lysate with recombinant caspases, suggesting that p35 was a caspase-cleaved fragment of XRCC4 in apoptotic cell death. p60 was sensitive to treatment with phosphatase or wortmannin and was undetectable in M059J cells deficient in DNA-PKcs. However, p60 was found in ataxia-telangiectasia cells after irradiation. These results indicated p60 as a phosphorylated form of XRCC4, requiring DNA-PKcs but not ataxia-telangiectasia mutated (ATM).

Therapeutic trials in muscular dystrophy. III. Studies of microbial proteinase inhibitors in murine dystrophy.

The microbial antiproteinases--antipain, leupeptin, and pepstatin--have been reported to inhibit the degeneration of chicken dystrophic muscle in tissue culture. Trials of antipain and pepstatin, and of leupeptin and pepstatin administered subcutaneously in murine muscular dystrophy, failed to produce evidence of benefit. It is suggested that these antiproteinases cannot pass through an intact sarcolemma into muscle fibers. Further studies with liposomes may allow these agents to enter muscle fibers.

Involvement of SAPK/JNK pathway in X-ray-induced rapid cell death of human T-cell leukemia cell line MOLT-4.

We found that SAPK/JNK was phosphorylated during X-ray-induced rapid cell death of MOLT-4 cells and that acid Sphingomyelinase inhibitor D609 suppressed the rapid cell death as well as phosphorylation of SAPK/JNK. Also C2-ceramide caused phosphorylation of SAPK/JNK, followed by rapid cell death. Further we isolated X-ray-resistant radiation-hybrid clones from MOLT-4 and 50 Gy irradiated mouse FM3A cells by repeated selections with 3 Gy irradiation. One of them named Rh-1a was found resistant to X-ray- as well as C2-ceramide-induced rapid cell death. Rh-1a cells had mouse DNA but no increase in either mouse or human Bcl-2 determined by Western blotting. Accumulation of p53 after X-irradiation was similarly observed in both parental MOLT-4 and Rh-1a cells. However, contrasting to prolonged and prominent phosphorylated status of SAPK/JNK in MOLT-4 cells, Rh-1a cells exhibited short transient increase and FM3A cells showed no increase of phosphorylated status SAPK/JNK after X-irradiation. Therefore, SAPK/JNK activation is considered important in X-ray-induced rapid cell death or apoptosis of MOLT-4 cells.

Monoclonal antibodies against hen's egg ovomucoid.

Two monoclonal antibodies (mAb 23E5 and 32A8) to hen's egg ovomucoid (OM), which causes hen's egg allergy and has trypsin inhibitory activity, were prepared and purified. Their affinity to the three separate domains of the ovomucoid, which are homologous in primary structure and are designated as DI, DII, and DIII, was studied by a competitive radioimmunoassay. MAb 23E5 bound to OM more efficiently than to DI, DII, or DIII-2 (with carbohydrate), but reacted with DIII-1 (free from carbohydrate) more efficiently than with OM. Except for the binding to OM, mAb 32A8 bound to DIII-2 most efficiently and to DIII-1 least efficiently, suggesting that this antibody recognized the carbohydrate moiety of DIII. MAb 32A8 inhibited the trypsin inhibitory activity of OM, whereas mAb 23E5 had no effect on it. These monoclonal antibodies should be useful for analyzing the antigenic determinants and trypsin inhibitory activity of ovomucoid.

High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes.

Nrf2, which belongs to the basic leucine zipper (bZip) transcription factor family, has been implicated as a key molecule involved in antioxidant-responsive element (ARE)-mediated gene expression. In order to examine the role of Nrf2 in protection against xenobiotic toxicity, the sensitivity of nrf2 knockout mice to acetaminophen (N-acetyl-4-aminophenol (APAP)) was analyzed. The saturation of detoxification pathways after high levels of exposure to APAP is known to induce hepatotoxicity. Two factors important in its detoxification are UDP-glucuronosyltransferase (UDP-GT), an ARE-regulated phase-II drug-metabolizing enzyme, and glutathione (GSH), an antioxidant molecule whose synthesis depends on ARE-regulated gamma-glutamylcysteine synthetase (gammaGCS). Two- to 4-month-old male mice were orally administered a single dose of APAP at 0, 150, 300, or 600 mg/kg. Doses of 300 mg/kg APAP or greater caused death in the homozygous knockout mice only, and those that survived showed a greater severity in hepatic damage than the wild-type mice, as demonstrated by increased plasma alanine aminotransferase activity, decreased hepatic non-protein sulfhydryl (NPSH) content, and centrilobular hepatocellular necrosis. The high sensitivity of Nrf2-deficient mice was confirmed from observations made at 0, 2, 8, and 24 h after dosing with 300 mg/kg APAP; increased anti-APAP immunoreactivity was also noted in their livers at 2 h. Untreated homozygous knockout mice showed both a lower UDP-GT activity and NPSH content, which corresponded to decreased mRNA levels of UDP-GT (Ugt1a6) and the heavy chain of gammaGCS, respectively. These results show that Nrf2 plays a protective role against APAP hepatotoxicity by regulating both drug metabolizing enzymes and antioxidant genes through the ARE.

Effect of serotonin (5-HT) on trigeminal rhythmic activities generated in in vitro brainstem block preparations.

We used rat isolated brainstem block preparations to analyze the functional roles of serotonin receptors in the generation of trigeminal rhythmic activities. We previously reported that trigeminal rhythmic activities could be induced by some pharmacological applications in an isolated brainstem preparation with a rostral boundary at the border between the inferior and superior colliculus, and a caudal border at the level of the rostral facial nucleus. However, the same stimulation did not induce trigeminal rhythmic activities in a whole brainstem block preparation with the same rostral boundary and a caudal border at the obex level. In the present study, both the 5-HT(1A) phthalimido-butyl-piperazine, and the 5-HT(2C) agonist, 1-2,5-dimethoxy-4-iodophenyl-2-aminopropane, combined with N-methyl-D,L-aspartate and bicuculline, elicited trigeminal rhythmic activities in a whole brainstem block preparation. Our results suggest that serotonin has both facilitation and inhibition effects on the generation of trigeminal rhythmic activities in an isolated brainstem block preparation in vitro.

Monoclonal antibody raised against tetrodonic acid, a derivative of tetrodotoxin.

Tetrodonic acid, a relatively non-toxic derivative of tetrodotoxin, was conjugated with bovine serum albumin and injected i.p. to BALB/c mice. After several injections, spleen cells were isolated, fused with myeloma cells X63-Ag8-6.5.3. and cloned by the limiting dilution method. The monoclonal antibody produced in ascites fluid in the mouse by the cloned cell showed an increasing reactivity with tetrodotoxin at concentrations ranging from 0.03 to 100 micrograms per well.

Epiphyseal lesions of the femur and tibia in rats following oral chronic administration of zinc dimethyldithiocarbamate (ziram).

A 24-month chronic feeding toxicity study with zinc dimethyldithiocarbamate (ziram) was performed on Fischer 344 rats of both sexes (80 animals/sex per group) at dietary levels of 0, 20, 200, or 2000 ppm. Eight animals of either sex from each group were sacrificed after 26, 52 and 78 weeks, and all surviving animals were killed after 104 weeks. Epiphyseal abnormalities in the long bones of the hind legs were observed in both sexes at 2000 ppm. Clinically, 3 male rats showed partial paralysis of the hind legs. At necropsy, marked curvature of the proximal end of the crus which could cause a restricted extension of the tibio-femoral joint was seen in 11 of 34 males killed at terminal sacrifice. While females had neither clinical signs nor gross abnormalities during the study, histopathological examination revealed retarded epiphyseal closure of the proximal end of the tibia in both sexes. Females also showed the epiphyseal lesion at the distal end of the femur. In severely affected rats marked proliferation of epiphyseal cartilaginous tissue was also noted together with the irregular arrangement of chondrocytes. These changes were evident only in aged animals. The incidence of the lesions in all males and females examined in this group was 22/77 (29%) and 13/73 (18%), respectively. The occurrence of the lesions appeared to be caused by impaired regulation of epiphyseal closure which might be related to the treatment with ziram.

Roles of DNA-dependent protein kinase and ATM in cell-cycle-dependent radiation sensitivity in human cells.

PURPOSE: The roles of DNA-dependent protein kinase (DNA-PK) and ATM in the cell-cycle-dependent radiosensitivity in human cells were investigated. METHODS AND MATERIALS: A DNA-PK activity-deficient human glioblastoma cell line M059J, ataxia telangiectasia cell lines AT3BISV and AT5BIVA, and control cell lines were used. Wortmannin inhibited DNA-PK and ATM activities. Cells were synchronized by hydroxyurea. Progression through the cell cycle was analysed by flow cytometry. RESULTS: M059J exhibited hyper-radiosensitivity throughout the cell cycle, with extreme hyper-radiosensitivity in G to early S-phase compared with the control cell line M059K. AT3BISV and AT5BIVA exhibited hyper-radiosensitivity throughout the cell cycle but showed a similar pattern of cell-cycle-dependent radiosensitivity to that observed in LM217 or HeLa cells. In AT3BISV and AT5BIVA, radiosensitization by wortmannin was observed throughout the cell cycle and was most prominent in G1 to early S-phase. Wortmannin did not sensitize M059J to ionizing radiation in any cell-cycle phase. DNA-PK activities were not different throughout the cell cycle. CONCLUSION: The results suggest that (1) non-homologous endjoining plays a dominant role in G1 to early S-phase and a minor role in late S to G2-phase in repairing DNA double-strand breaks, (2) the role of ATM in repairing double-strand breaks may be almost cell-cycle-independent and (3) the dominant role of non-homologous end-joining during G1 to early S-phase is not due to cell-cycle-dependent fluctuations in DNA-PK activity.

Decreased c-Myc expression and its involvement in X-ray-induced apoptotic cell death of human T-cell leukaemia cell line MOLT-4.

PURPOSE: To investigate the possible involvement of c-Myc and ceramide-c-Jun N-terminal kinase (JNK) pathway in X-ray-induced apoptotic cell death of MOLT-4 cells. MATERIALS AND METHODS: The expressions of c-Myc protein and c-myc mRNA after X-irradiation were analysed by Western blotting and RT-PCR between radiosensitive MOLT-4 and radioresistant variant Rh-1a cells with less JNK activation than the parental cells. Apoptotic cell death was determined by a dye exclusion test, the appearance of chromatin condensation and DNA fragmentation. The effect of a JNK activator anisomycin or c-Myc inhibitor peptides (Int-H1-S6A, F8A) on the amount of c-Myc protein and on the induction of apoptosis was investigated, respectively. RESULTS: In X-irradiated MOLT-4 cells, amounts of both c-myc mRNA and c-Myc protein rapidly decreased, which was followed by apoptotic cell death, while little change or limited reduction of c-Myc protein was observed in X-irradiated Rh-1a cells with accompanying higher cell viability. Exposure of MOLT-4 and Rh-1a cells to c-Myc inhibitor peptides similarly induced apoptotic cell death with decreases of c-Myc protein. Anisomycin rapidly induced JNK activation and a subsequent decrease of c-Myc protein, causing cell death in MOLT-4 cells. On the other hand, Rh-1a cells were more resistant to anisomycin than parental MOLT-4 cells, showing less JNK activation and a delayed decrease of c-Myc protein. CONCLUSION: A decrease of c-Myc protein was considered important in X-ray-induced apoptotic cell death of MOLT-4 cells; activation of the JNK pathway caused reduction in the amounts of c-myc mRNA and c-Myc protein, and finally induced apoptotic cell death.