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Aneurysmal bone cyst is a benign cystic lesion of bone, and the incidence of this disease in the jaw is low. We reported two cases of aneurysmal bone cyst of the jaw. A 31-year-old woman presented with complaints of pain and swelling at the left side of the face. Since X-ray and CT-scan suggested a cystic lesion, cystectomy was performed. The lesion was histologically diagnosed to be an aneurysmal bone cyst. The other patient, a 40-year-old man, showed swelling at the right mandible. The patient's occlusion was impaired by the gingival swelling. X-ray findings showed the presence of a honeycomb-like lesion at the body and ramus of the mandible. CT-scan findings showed buccal and lingual expansion at the molar region. Microscopic features of the biopsy specimen were consistent with diagnosis of an aneurysmal bone cyst. We performed conservative curettage of the lesion. The postoperative courses of both of these cases were uneventful, and there has been no sign of recurrence after 7 years and 4 years, respectively.

The treatment of tetrapod zygomatic fractures is generally preceded by open reduction and fixation by means of supraorbital eyebrow and infraorbital incisions. However, fixation of the infraorbital rim does not produce adequate stability of the fractured zygoma owing to the thinness of this bone. We used open reduction and fixation via a supraorbital eyebrow incision and an intraoral approach in three cases of zygomatic fracture. The advantage of this intraoral approach is that it allows reduction under direct vision and provides sufficient stability after mini-plate fixation. Consequently, satisfactory results were obtained.

Osteosarcoma of jaws does not always have characteristic clinical symptoms or X-ray findings. Moreover, the disease is sometimes difficult to diagnose histopathologically even based on biopsy specimens. Here, two cases of osteosarcoma of the mandible, which were difficult to diagnose definitely, are reported.Case: A 38-year-old female complained of paresthesia of the mental region. Osteomyelitis was suspected on clinical examination. A biopsy was performed, but a definitive diagnosis could not be established. Subsequently, the swelling increased and the radiological examination showed a ‘sun-ray’ appearance. On histopathological re-examination, a diagnosis of osteosarcoma was established.Case 2: A 38-year old male complained of paresthesia of the left lower lip and swelling of the buccal gingiva of the left lower premolar region. A biopsy was performed under a clinical diagnosis of a malignant tumor of the mandible. However, the histological diagnosis was an aneurysmal bone cyst. After total enucleation, a diagnosis of osteosarcoma was histopathologically established.

Calcifying epithelioma is a tumor originating in hair cortex cells. It tends to occur in the head and neck region.We reported a case of rapidly growing calcifying epithelioma. The patient, a 12-yearold girl, had a bone-like hard tumor in her parotidomasseteric region. The tumor had grown rapidly for the last three months before operation. The tumor was extirpated and histological examination revealed a calcifying epithelioma. An inflammatory stimulus apparently promoted the rapid growth of this tumor. There has been no evidence of recurrence for 8 years after operation.

Paclitaxel (Taxol, TXL) is an antimicrotubule agent that stabilizes microtubules, arrests the cell cycle at the G(2)/M phase and induces apoptosis. In vitro drug sensitivity assays have shown that the combination of TXL and CDDP is more effective in CDDP-resistant ovarian carcinoma cell lines, with different cytotoxicities depending on the sequence of drug exposure. CDDP also shows poor results in human epidermoid carcinoma particularly of the head and neck region.We investigated the effects and the molecular mechanisms of combination chemotherapy with TXL and CDDP in the CDDP-resistant cell line A431/CDDP2, and in its parental human epidermoid cell line A431/P. Drug sensitivity was determined using the MTS assay and cell cycle perturbation was analyzed using flow cytometry. DNA fragmentation was then analyzed and the protein levels of caspase-3 and Bcl-2, and phosphorylated of Bcl-2 were determined by Western blotting.In the drug sensitivity assay, exposure to CDDP prior to TXL was more effective than exposure TXL prior to CDDP in A431/P cells. In A431/CDDP2 cells, exposure to TXL prior to CDDP was more effective than exposure to CDDP prior to TXL. Exposure to TXL arrested the cells in the G(2)/M phase in both cell lines. In A431/CDDP2 cells, exposure to TXL prior to CDDP arrested the cells in the G(2)/M phase, an effect caused by either CDDP or TXL. Analysis of DNA fragmentation showed similar results to the drug sensitivity assay. Expression of caspase-3 protein active form was detected following exposure to TXL only and to the TXL/CDDP combination in both A431/P and A431/CDDP2 cells, but phosphorylation of Bcl-2 protein was detected only following exposure to TXL and only in A431/CDDP2 cells.These results indicate that exposure to TXL prior to CDDP plays a key role in circumventing CDDP resistance by phosphorylating Bcl-2 protein in the human epidermoid carcinoma cell line A431/CDDP2.

Objective. The purpose of this study was to compare odontogenic keratocysts associated with and not associated with an impacted mandibular third molar. Study Design. Panoramic radiographs of odontogenic keratocysts associated with impacted mandibular third molars (15 cysts, associated group) were compared with radiographs of odontogenic keratocysts not associated with impacted mandibular third molars (25 cysts, nonassociated group). The radiographic images were analyzed with reference to the patients' age and sex. Results. The mean age of patients in the associated group was lower than that of patients in the nonassociated group. The mean area of the cysts in the associated group was larger than that of those in the nonassociated group. The patients' ages did not significantly correlate to the areas of either kind of cyst. Conclusions. The odontogenic keratocysts in the associated group had a tendency toward rapid growth in the patients' youth. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:272-5)

A 33-year-old man presented with Kimura's disease of the hard palate. Serological examination showed no elevation in the number of eosinophils or in IgE level, but histological examination revealed well-developed lymph follicles with increased numbers of eosinophils. In addition, immunohistochemical examination revealed that the IgE concentrations were shown in the lymphoid follicles with germinal centers, a finding typical of Kimura's disease. The patient underwent surgical resection followed by treatment with non-steroidal anti-inflammatory drugs (NSAIDs). Throughout the 2-year follow-up period, the patient's condition has remained satisfactory.

Cyclin-dependent kinases (Cdks) play essential roles in the intracellular controls of the cell cycles. Roscovitine, [2-(R)-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine], is a potent and selective inhibitor of the Cdk2 and Cdc2. We investigated whether this compound was effective against head and neck squamous cell carcinoma (HNSCC) cells. Roscovitine was found to inhibit the growth of all 11 HNSCC cell lines in time- and dose-dependent manner and to diminish the Cdk2 and Cdc2 activities. An induction of apoptosis was observed in all cells, as judged by the cell morphology, along with the appearance of cells with sub-G 1 DNA contents, DNA fragmentations, and poly (ADP-ribose) polymerase (PARP) cleavage. In four HNSCC cell lines, apoptosis was induced without antecedent marked cell cycle arrest, and in the other seven cell lines, cell cycle arrest preceded cell death. We also found up-regulation of Bcl-x s in the former cell lines, and in the latter cell lines, the expressions of Bcl-2 and Bcl-x L were induced simultaneously. These results suggest that roscovitine exerts antitumor activities in HNSCC and is associated with induction of Bcl-x S . Roscovitine can be considered to provide a new chemopreventive and chemotherapeutic strategy for the clinical management of HNSCC.

We previously reported a blast coating method (BC method) as a new coating method of titanium (Ti) with apatite (AP) at room temperature. The BC method gives much stronger AP coating on the Ti surface compared with those obtained by other room temperature coating methods. However, no in vivo study has been made, so far, to evaluate the stability or the tissue response to the implant. As an initial step to evaluate the feasibility of the BC method, we evaluated the tissue response and stability of AP coated Ti implant prepared with the BC method (AP–BC implant) using rats as experimental animals. The AP coating adhered tightly to the Ti surface even after the implant procedure and throughout the experimental period up to six weeks post operation. AP–BC implant caused no inflammatory response, showed strong bone response and much better osteoconductivity compared with the pure Ti implant. The new bone formed on the surface of AP–BC implants was thinner compared with that formed on the surface of Ti implant. Therefore, the AP–BC implant has a good potential as an osteoconductive implant material.

Heat shock proteins (HSPs) have been defined as proteins induced by heat shock and other environmental and pathophysiologic stress. Heat shock protein 27 (HSP27) is one of the small heat shock proteins. HSP27 is implicated in protein-protein interactions such as folding, translocation, and prevention of inappropriate protein aggregation. Many of their functions suggest that they play important roles in cancers. Archival tissues from 40 patients with oral squamous cell carcinoma who received primary surgical resection were examined for HSP27 by immunohistochemistry and correlated with clinical stage, lymph node metastasis, histological grade and survival period. HSP27 expression was positive staining (+) in 20 (50%), weak or negative staining (-) in 20 (50%) of total 40 cases. There was no correlation between HSP27 expression and clinical stage, lymph node metastasis and histological grade. However, when compared with clinicopathological features, the expression of HSP27 correlated inversely with survival period. This study suggests that the expression of HSP27 is frequently promoted in patients with oral squamous cell carcinoma and should be considered an independent prognostic factor of oral squamous cell carcinoma patients.

To evaluate the role of several specialized mechanisms for D-glucose transport in human oral mucosa, a cultured stratified cell layer derived from human oral mucosa was employed. Although this culture system has been used for reconstructive surgery, we, for the first time, tried to apply this system to the evaluation of nutrients and drug transport. Cell number and transepithelial electrical resistance (TEER) reached steady state 7–8 days after inoculation on the Transwell and TEER values at steady state were 130–140 Ω cm2, which was higher or lower than that of small intestine or Caco-2 cells, respectively. The transport studies were carried out using the cultured epithelium on the Transwell. The transport of D-glucose across the cultured stratified layer of oral epithelial cells was much more extensive than L-glucose, and was inhibited by 2-deoxy-D-glucose, a substrate of facilitative glucose transporters, and α-methyl-D-glucoside, a specific substrate of a Na+/glucose cotransporter (SGLT1). The results indicate that the sugar transporters function not only to take up D-glucose by the epithelial cells but also to transport the sugar across the stratified epithelial layer.

Connective tissue growth factor (CTGF) is a potent secreted signaling factor which functions in multiple stages of angiogenesis. In the present study, we examined the role of CTGF in tumor angiogenesis and made the following observations: (1) Histological analysis of human breast cancer (MDA231) cell and human fibrosarcoma (HT1080) cell xenografts in BALB/c nude mice showed a high level of neovascularization. Human squamous cell carcinoma (A431) xenografts induced only a low level of neovascularization. (2) CTGF mRNA was strongly expressed in MDA231 and in HT1080 cells in vivo and in vitro, but not in A431 cells. (3) CTGF protein was markedly produced in MDA231 cells and HT1080 cells and secreted into culture medium, and its production was greater during phases of growth rather than confluency. (4) Production of CTGF in bovine aorta endothelial cells was induced by CTGF, VEGF, bFGF and TGF-β. (5) Neovascularization induced by HT1080 cells or MDA231 cells on chicken chorioallantoic membrane was suppressed in the presence of neutralizing CTGF-specific polyclonal antibody. These results suggest that CTGF regulates progression in tumor angiogenesis and the release or secretion of CTGF from tumor cells is essential for the angiogenesis.

Reduction of E-cadherin strongly relates to invasiveness and metastasis in vitro. To clarify CpG methylation around the promoter region of the E-cadherin gene in oral squamous cell carcinoma (SCC), we examined the DNA samples of various human SCC cell lines and primary oral SCC tissues by methylation-specific polymerase chain reaction (MSP). CpG methylation of the E-cadherin gene markedly correlated to the reduction of E-cadherin expression in human oral SCC cell lines. In primary oral SCC tissues, only 1 of 5 preserved E-cadherin-expressing tissues was methylated, whereas methylation was found in 17 (94.4%) of 18 E-cadherin-reduced tissues. Our results suggest that reduction of E-cadherin expression is associated with CpG methylation of the E-cadherin gene promoter. We recently established two cell lines with high and low metastatic potential, UM1 and UM2, from SCC primary tongue tissue of a patient. E-cadherin expression of high-metastatic UM1 was clearly lower than that of low-metastatic UM2, and MSP results showed CpG methylation in the UM1 but not the UM2 cell line. To investigate whether demethylation of CpG methylation of the E-cadherin gene could restore expression and function of E-cadherin, we treated UM1 with the demethylating agent 5-azacytidine (5-aza) and found that E-cadherin expression was indeed restored by demethylation. Moreover, in the demethylated UM1, invasion of the collagen gel was clearly suppressed compared with the untreated UM1. These results suggested that inactivation of E-cadherin expression resulted from CpG methylation of the gene promoter; a correlation between CpG methylation of the E-cadherin gene promoter and invasive potential was also suggested.

The p14(ARF), p15(INK4B) and p16(INK4A) genes were localized to 9p21, where genetic alterations have been reported frequently in various human tumors. We performed a molecular analysis of the mechanism of inactivation in cell lines and 32 oral squamous cell carcinoma (OSCC), using deletion screening, PCR-SSCP, methylation-specific-PCR and cycle sequencing. We detected homozygous deletion of p14(ARF)-1Ebeta in 9 (26.5%), of p15(INK4B) in one (3.1%), and of p16(INK4A) in 22 (56.3%) tumor samples. Three mutations were detected in the p16(INK4A) genes. We detected aberrant methylation of the p14(ARF) genes in 14 (43.8%), of the p15(INK4B) gene in 9 (28.1%), and of the p16(INK4A) gene in 16 (50.0%) tumor samples. Altogether, 87.5% of the samples harbored at least one of the alterations in the p14(ARF), p15(INK4B), and p16(INK4A) genes, indicating that the frequent inactivation of these genes may be an important mechanism during OSCC development.

Drosophila ma-l gene was suggested to encode an enzyme for sulfuration of the desulfo molybdenum cofactor for xanthine dehydrogenase (XDH) and aldehyde oxidase (AO). The human molybdenum cofactor sulfurase (HMCS) gene, the human ma-l homologue, is therefore a candidate gene responsible for classical xanthinuria type II, which involves both XDH and AO deficiencies. However, HMCS has not been identified as yet. In this study, we cloned the HMCS gene from a cDNA library prepared from liver. In two independent patients with classical xanthinuria type II, we identified a C to T base substitution at nucleotide 1255 in the HMCS gene that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 419. A classical xanthinuria type I patient and healthy volunteers lacked this mutation. These results indicate that a functional defect of the HMCS gene is responsible for classical xanthinuria type II, and that HMCS protein functions to provide a sulfur atom for the molybdenum cofactor of XDH and AO.

Background: Telomerase is a ribonucleoprotein enzyme which is involved in the maintenance of chromosome ends. Telomerase activity is frequently associated with malignant phenotypes, and it can be considered a ubiquitous tumor marker. In this study we describe an approach for developing in vitro chemosensitivity assays based on the assessment of telomerase activity in squamous cell carcinoma to treatment with anticancer drugs. Methods: We used A431/CDDP1 and A431/CDDP2, two previously established cisplatin (CDDP)-resistant A431 sublines. These cell lines were not multidrug resistant but specifically resistant to the CDDP drug family. The telomeric repeat amplification protocol (TRAP assay) was used to measure telomerase activity in CDDP-resistant cell lines and to examine the effect of CDDP and other anticancer drugs on enzyme levels. We next analyzed the relations between telomerase activity and cytotoxic effects of CDDP in human squamous cell carcinoma (HSC2, HSC3, HSC4 and KB cell lines). Results: The telomerase activity of A431/CDDP1 and A431/CDDP2 was significantly higher than that of the parent A431 cell (A431/P) treated with CDDP and carboplatin at IC50. On the other hand, telomerase activity in these cell lines was not influenced by treatment with 5-fluorouracil, bleomycin, Adriamycin or taxol. The regression line derived from the quantitative analysis of the telomeric ladder versus IC50 of CDDP concentrations was analyzed. Telomerase activity was found to be positively correlated with the IC50 values for CDDP. Conclusions: The results of the present studies on in vitro squamous cell carcinoma cell lines indicate that telomerase activity inhibition can be used as a marker of tumor cell killing by CDDP. Therefore, the present investigation provides a rational basis for an in vitro chemosensitivity assay based on telomerase activity evaluation.

The relationship between clinicopathological factors and response of radiation therapy in oral squamous cell carcinoma has been studied. It has been suggested that factors such as tumor site, extent and tumor differentiation determine the response to radiation therapy. It is known that oxygenation is related to the therapeutic effects of radiation therapy. However, there are few reports on the relationship between oxygen condition and the response to radiation therapy. The present study was carried out to assess whether any clinicopathological factors, including an evaluation of the oxygen condition can be used to predict the effects of preoperative radiation therapy in oral squamous cell carcinomas. Forty-seven patients with oral cancer treated with external radiation therapy preoperatively were evaluated. There were no significant differences in response to the radiation with respect to age, sex, tumor site, stage, macroscopic shape of tumors, and the histological factors. The hemoglobin (Hb) and arterial oxygen content (CaO 2 ) levels of favorable cases (Hb: 14.4 g/dl, CaO 2 19.1 ml/dl) were significantly higher than those of unfavorable cases (Hb: 11.0 g/dl, CaO 2 : 16.1 ml/dl). These findings suggest that oxygen conditions of oral cancer patients predict tumor response to preoperative radiation therapy.

Immunohistochemical analysis of Rb, p16(INK4A) and cyclin D1 expression was performed on 78 oral squamous cell carcinoma (SCC), 46 leukoplakia, and 20 normal mucosa. Rb and p16(INK4A) expression were observed in all normal mucosa and most of leukoplakia. Lack of Rb and p16(INK4A) was observed in 56.4 and 67.9% of SCC, respectively. The overexpression of cyclin D1 was not observed in normal mucosa and was observed in 35.9% of SCC. A strong reciprocal relationship between Rb and p16(INK4A) expression was observed in oral SCC, and all these SCC cases have at least one of the alterations in the Rb pathway.

Epidermal growth factor receptor (EGFR) frequently overexpresses in cancers, including oral squamous cell carcinomas (OSCC). We previously identified a truncated EGFR (tEGFR) in human oral keratinocytes. In this study, we evaluated the prognostic value of tEGFR in 45 cases of OSCC. tEGFR expression inversely correlated with EGFR expression (r=-0.83, P

The mechanism of induction of emesis by X-ray irradiation remains largely unknown. The purpose of the present research was to clarify the neuronal basis of the induction of nausea induced by X-ray irradiation analyzing c-Fos expression in the nucleus tractus solitarii (NTS) as a marker of cellular excitation. We confirmed that the dose of X-ray irradiation (4 Gy) used for the present research could actually induce nausea by preliminary measurement of kaolin intake. Induction of c-Fos immunoreactivity in the NTS was observed in the animals that received X-ray irradiation of the whole body. The mean number of c-Fos positive cells in the animals that received irradiation was significantly larger than that in the non-irradiated animals. Partial exposure of the abdomen to X-rays showed significantly greater c-Fos expression than that of the head. These results indicated the presence of a certain route for transmitting information from the periphery toward the central nervous system by X-ray irradiation. The number of c-Fos positive cells induced by X-ray irradiation in animals vagotomized at the subdiaphragmatic level was lower than that in sham-operated animals. Animals receiving a serotonin subtype three (5-HT3, 5-hydroxytryptamine) receptor antagonist (tropisetron, ICS 205-930, 3-tropanyl-indole-3-carboxylate) showed a significant reduction in c-Fos protein expression compared to animals receiving a vehicle. These results strongly suggested that X-ray irradiation activates 5-HT3 receptors on the terminals of the abdominal vagal nerves to excite the afferent pathway, thereby inducing emesis.

In the C 4 plant maize ( Zea mays L.), two ferredoxin isoproteins, Fd I and Fd II, are expressed specifically in mesophyll and bundle‐sheath cells, respectively. cDNAs for these ferredoxins were introduced separately into the cyanobacterium Plectonema boryanum with a disrupted endogenous ferredoxin gene, yielding TM202 and KM2‐9 strains expressing Fd I and Fd II. The growth of TM202 was retarded under high light (130 μmol/m 2 /s), whereas KM2‐9 grew at a normal rate but exhibited a nitrogen‐deficient phenotype. Measurement of photosynthetic O 2 evolution revealed that the reducing power was not efficiently partitioned into nitrogen assimilation in KM2‐9. After starvation of the cells in darkness, the P700 oxidation level under far‐red illumination increased significantly in TM202. However, it remained low in KM2‐9, indicating an active cyclic electron flow. In accordance with this, the cellular ratio of ATP/ADP increased and that of NADPH/NADP + decreased in KM2‐9 as compared with TM202. These results demonstrated that the two cell type‐specific ferredoxins differentially modulate electron flow around photosystem I.

Radical scavengers play an important role in cancer cells defending themselves against free radicals which occur with irradiation. SOD (Cu,Zn, Mn-) and GST-pi are radical scavengers with an effect on radiation therapy. We investigated the correlation between radiation effects and expression of Cu,Zn-, Mn-SOD and GST-pi in 34 cases of oral cancer, treated with preoperative radiation therapy. In this study, 22 cases out of 34 were classified as effective and 12 cases as non-effective. Expression of Cu,Zn, Mn-SOD and GST-pi were observed in 13 (38.2%), 10 (29.4%) and 20 (58.8%) cases, respectively. Regarding the value of radiation sensitivity from expression of these proteins in the biopsy samples, no significant correlation was found between those expressions and histological effectiveness of preoperative radiation therapy. But interestingly, in 11 out of 12 of the non-effective cases, strong staining of Cu, Zn-SOD and GST-pi were shown at the residual cancer cells after preoperative radiation therapy. These results suggested that the expression of SOD (Cu,Zn-, and Mn-) and GST-pi may be not useful markers for predicting the effects of radiation therapy. However, Cu, Zn-SOD and GST-pi were increased by irradiation and may play an important role in radiation resistance and cancer cell regeneration after radiation therapy.

Lateral soft tissue cephalometric standards of Japanese normal adults were developed with the use of Ricketts, Epker, Holdaway, and Legan cephalometric analyses. Cephalometric radiographs of 211 Japanese normal adults were analyzed, and the soft tissue measurements were compared with those of an esthetically pleasant Japanese subgroup and white adult sample. Statistically significant differences were found in the Japanese sample when compared with the white norms. On the other hand, the soft tissue mean values of the Japanese supernormal group were similar to the white norms, with the exception of the nasolabial angle and the lip prominence. Soft tissue cephalometric norms are specific for ethnic groups, but these values should not be interpreted as treatment goals. Normative data represent an aid for the diagnosis and planning of orthodontic treatment and orthognathic surgery.

In higher plants ferredoxin (Fd):NADP+ oxidoreductase (FNR) and Fd are each distributed in photosynthetic and non-photosynthetic organs as distinct isoproteins. We have cloned cDNAs for leaf FNR (L-FNR I and L-FNR II) and root FNR (R-FNR) from maize (Zea mays L.), and produced recombinant L-FNR I and R-FNR to study their enzymatic functions through kinetic and Fd-binding analyses. The K m value obtained by assay for a diaphorase activity indicated that R-FNR had a 10-fold higher affinity for NADPH than L-FNR I. When we assayed for NADPH-cytochromec reductase activity using maize photosynthetic Fd (Fd I) and non-photosynthetic Fd (Fd III), the R-FNR showed a marked difference in affinity between these two Fd isoproteins; theK m for Fd III was 3.0 μm and that for Fd I was 29 μm. Consistent with this, the dissociation constant for the R-FNR:Fd III complex was 10-fold smaller than that of the R-FNR:Fd I complex. This differential binding capacity was confirmed by an affinity chromatography of R-FNR on Fd-sepharose with stronger binding to Fd III. L-FNR I showed no such differential interaction with Fd I and Fd III. These data demonstrated that R-FNR has the ability to discriminate between these two types of Fds. We propose that the stronger interaction of R-FNR with Fd III is crucial for an efficient electron flux of NADPH-FNR-Fd cascade, thus supporting Fd-dependent metabolism in non-photosynthetic organs.

The hamster ornithine decarboxylase antizyme (ODC-Az) cDNA was transfected into the hamster malignant oral keratinocyte cell line, HCPC-1. Ectopic expression of ODC-Az resulted in the reversion of malignant phenotypes and alteration of DNA methylation status of CCGG sites. The phenotypes examined include ODC enzymatic activity, doubling time, morphological change, anchorage dependent growth, tumorigenicity in nude mice, induction of epithelial differentiation marker protein (involucrin), and change of cell cycle position. Comparison of CCGG DNA methylation status of the ODC-Az and control vector transfectants revealed a significant increase in demethylation of 5-methyl cytosines (m5C) of CCGG sites in the ODC-Az transfectants. Ectopic expression of ODC-Az gene in hamster malignant oral keratinocytes led to reduce ODC activity and the subsequent demethylation of 5-methyl cytosines, presumably via the ODC/ polyamines/ decarboxylated S-adenosylmethionine (dc-AdoMet) pathways. Our data suggest that ODC-Az shared the same pathway of polyamines/ dc-AdoMet/DNA methyltransferase (DNA MTase). We propose that ODC-Az mediates a novel mechanism in tumor suppression by DNA demethylation and presumably re-activation of key cellular genes silenced by DNA hypermethylation during cancer development. Oncogene (2001) 20, 24 - 33.

There are still no objective diagnostic criteria for macroglossia. The aims of this study were to examine the effect of reduction of the tongue on its position, and to suggest a standard measurement point for the objective diagnosis of macroglossia. Twenty-four patients were studied. Lateral cephalometric radiographs were taken with the tongue in the rest position, and preoperative and postoperative measurements were made on cephalometric lateral roentgenograms from all 14 patients with macroglossia and the 10 control subjects. The results suggested that measurement of length of the tongue, and the tongue area: oral cavity ratio on lateral cephalometric radiographs are useful in the more accurate diagnosis of macroglossia.

Cisplatin (cis-diamminedichloroplatinum(II), CDDP) is one of the most important chemotherapeutic agents; however, the mechanisms of resistance to this drug are still unknown. Recent reports have demonstrated that chemotherapy can induce apoptosis in some cancer cells, indicating that apoptosis may play a very important role in cancer therapy. Therefore, we used a CDDP-resistant cell line from the human epidermoid carcinoma cell line A431 to investigate whether the modulation of apoptosis influences CDDP resistance. In the CDDP-resistant cell, the cell cycle was not perturbed after CDDP treatment. DNA gel electrophoresis and ELISA of the CDDP-resistant cell showed reduced apoptosis when compared with A431 cells treated with CDDP. We determined the p53, Bcl-2, Bax and CPP32 protein levels by Western blotting. This analysis demonstrated a marked increase in Bcl-2 protein levels and a reduction in CPP32 protein levels in CDDP-resistant cells. Our results indicate that the reduction of apoptosis was one of the CDDP-resistant mechanisms, and that reduced apoptosis in CDDP-resistant cells was influenced by Bcl-2 and CPP32 proteins.

Metallothionein (MT), a low molecular weight metal-binding protein, has been related to zinc and copper metabolism, the acute-phase response, and cellular proliferation. In this study, we investigated changes in zinc metabolism and MT gene expression occurring in tissue damage and repair during wound healing in mouse skin. Northern blot analysis revealed that a significant increase of MT mRNA was observed in the liver for 18 h after wounding, and serum zinc downfall and hepatic zinc uptake were observed. In situ hybridization analysis showed that no significant expression of MT mRNA was detected within the first 9 h after wounding. However, it was expressed restrictively in the proliferating epidermis of the wound margin after 12 h. Zinc began to accumulate in wounded skin after MT gene expressed. Northern blotting and immunocytochemical staining revealed that MT has been synthesized actively during the growth phase compared with the stationary phase in normal human epidermal keratinocytes in vitro. Intracellular zinc accumulation was observed in the proliferating cells. We concluded that hepatic MT plays an important role as an acute phase protein against host damage, and epidermal MT contributes in the supply of zinc to wounded tissue and activates proliferation for the regeneration of epidermis.

Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) were found to inhibit strongly the citrulline formation activity of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39). Such inhibition was not observed with any other phospholipid examined. A kinetic analysis of purified nNOS showed no significant change in apparent K(m) for L-Arg or NADPH caused by these inhibitory phospholipids. Electron paramagnetic resonance analysis revealed no significant spectral perturbation of the ferriheme or flavin semiquinone upon the addition of PIP2. On the other hand, a lower enhancement of the NADPH diaphorase activity by Ca(2+)-calmodulin was observed in the presence of PIP2 and PA, and the citrulline formation activity was protected from phospholipid inhibition by preincubation with Ca(2+)-calmodulin. Moreover, trypsin digestion analysis showed that the cleavage site within the calmodulin-binding site of nNOS was specifically protected from trypsin by the addition of PIP2 and PA. These results strongly suggest that PIP2 and PA inhibit the citrulline formation activity of nNOS by blocking the interaction of the enzyme with Ca(2+)-calmodulin.

Plant-type ferredoxin (Fd), a [2Fe-2S] iron-sulfur protein, functions as an one-electron donor to Fd-NADP(+) reductase (FNR) or sulfite reductase (SiR), interacting electrostatically with them. In order to understand the protein-protein interaction between Fd and these two different enzymes, 10 acidic surface residues in maize Fd (isoform III), Asp-27, Glu-30, Asp-58, Asp-61, Asp-66/Asp-67, Glu-71/Glu-72, Asp-85, and Glu-93, were substituted with the corresponding amide residues by site-directed mutagenesis. The redox potentials of the mutated Fds were not markedly changed, except for E93Q, the redox potential of which was more positive by 67 mV than that of the wild type. Kinetic experiments showed that the mutations at Asp-66/Asp-67 and Glu-93 significantly affected electron transfer to the two enzymes. Interestingly, D66N/D67N was less efficient in the reaction with FNR than E93Q, whereas this relationship was reversed in the reaction with SiR. The static interaction of the mutant Fds with each the two enzymes was analyzed by gel filtration of a mixture of Fd and each enzyme, and by affinity chromatography on Fd-immobilized resins. The contributions of Asp-66/Asp-67 and Glu-93 were found to be most important for the binding to FNR and SiR, respectively, in accordance with the kinetic data. These results allowed us to map the acidic regions of Fd required for electron transfer and for binding to FNR and SiR and demonstrate that the interaction sites for the two enzymes are at least partly distinct.

Alginate membrane was proposed as a self-setting barrier membrane that can be used for guided tissue regeneration (GTR). The alginate membrane can be prepared and placed at the bone defect during the surgical procedure. The procedure consists of two simple steps. First, the bone defect is filled with sodium alginate (Na-Alg) aqueous solution. Then calcium chloride aqueous solution is dropped on the surface of the Na-Alg aqueous solution. An alginate membrane is formed on the bone defect, keeping the inside of the bone defect filled with unreacted Na-Alg aqueous solution. In this investigation, a preliminary animal study was conducted for an initial evaluation as to whether or not the alginate membrane can be used as a barrier membrane for the GTR method. Bone defects were made in the tibiae of 15-week-old rats. The alginate membrane was made on the surface of existing bone by filling the defect with Na-Alg aqueous solution and then dropping calcium chloride aqueous solution onto the surface of the Na-Alg solution. Four weeks after surgery, the bone defect was found to be reconstructed with new bone when the defect had been covered with alginate membrane whereas the bone defect was filled only with connective tissue when it had been kept open. We concluded, therefore, that this alginate membrane may be a useful barrier membrane when the GTR method is employed.

The setting reaction and mechanical strength in terms of diametral tensile strength (DTS) of hydroxyapatite (HAP) putty made of tetracalcium phosphate, dicalcium phosphate anhydrous, and neutral sodium hydrogen phosphate (Na1.8H1.2PO4) solution containing 8 wt % sodium alginate were evaluated as a function of the Na1.8H1.2PO4 concentration. In one condition, HAP putty was placed in an incubator kept at 37 degrees C and 100% relative humidity. In the other condition, immediately after mixing HAP putty was immersed in serum kept at 37 degrees C. Longer setting times and lower DTS values were observed when HAP putty was immersed in serum regardless of the Na1.8H1.2PO4 concentration. The setting times of the HAP putty in both conditions became shorter with an increase in the Na1. 8H1.2PO4 concentration, reaching approximately 7-13 min when the Na1. 8H1.2PO4 concentration was 0.6 mol/L or higher. The DTS value of HAP putty was relatively constant (10 MPa) regardless of the Na1.8H1. 2PO4 concentration (0.2-1.0 mol/L) when HAP putty was kept in an incubator. In contrast, when HAP putty was immersed in serum, the DTS value was dependent on the Na1.8H1.2PO4 concentration. It increased with the Na1.8H1.2PO4 concentration and reached approximately 5 MPa when the Na1.8H1.2PO4 concentration was 0.6 mol/L, after which it showed a relatively constant DTS value. We therefore would recommend a HAP putty that uses 0.6 mol/L Na1.8H1. 2PO4 since at that concentration the putty's setting time (approximately 10 min) is proper for clinical use and it shows good DTS value (approximately 5 MPa) even when it is immersed in serum immediately after mixing.

In the oxygenic photosynthetic organisms, ammonia is assimilated into glutamate through the combined actions of glutamine synthetase (GS) and glutamate synthase (glutamine 2-oxoglutarate amidotransferase or GOGAT). GS catalyzes the ATP-dependent amination of glutamate to yield glutamine. GOGAT catalyzes the reductive transfer of the amide group of glutamine to the keto position of 2-oxoglutarate to yield two molecules of glutamate. The GS/GOGAT pathway ultimately requires ATP and reducing power, which are generated by photosynthesis and oxidative catabolism of carbohydrates, and utilizes carbon skeletons. This pathway is thus involved in the integration of carbon and nitrogen assimilations.

Adenoid cystic carcinoma (ACC) is a salivary malignant tumor with poor long-term prognosis, that is known to have predilection for invasion of the adjacent stroma and neural tissues. This carcinoma has shown a high incidence of recurrence and distal metastasis. Invasive carcinomas have been associated with the distributions of extracellular matrices (ECM). Cell proliferation as a marker of tumor growth has been related to poor prognosis in oral carcinomas. Immunohistochemical analysis of 15 cases of ACC was done using antibodies to laminin, type IV collagen, fibronectin, tenascin and anti-proliferating nuclear antigen (PCNA). Laminin and type IV collagen were totally or partially absent in the ACC invasive areas. Tenascin was expressed in the stroma and cytoplasm and was associated with tumor cell proliferation. It can be concluded that basement membrane represents a barrier that is lost during cell invasion and tenascin may be involved in the detachment of cancer cells, increasing the invasive potential of ACC.

We have identified and characterized a nitrate-inducible ferredoxin (Fd) in maize (Zea mays L.) roots by structural analysis of the purified protein and by cloning of its cDNA and gene. In maize Fd isoproteins are encoded by a small multigene family, and the nitrate-inducible Fd was identified as a novel isoprotein, designated Fd VI, which differed from any Fd I to Fd V identified to date. In the roots of seedlings cultured without nitrate, Fd VI was undetectable. However, during the induction of the capacity for nitrate assimilation, the amount of Fd VI increased markedly within 24 h. Concurrently, the level of transcript for Fd VI increased, but more quickly, reaching a maximal level within 2 h with kinetics similar to those of nitrite reductase and Fd-NADP+ reductase. Fd III was constitutively expressed in roots, and no such changes at the protein and mRNA levels were observed during the nitrate induction. In the 5[prime] flanking region of the gene for Fd VI only, we identified NIT-2 motifs, which are widely found in genes for enzymes related to nitrogen metabolism. These data indicate that Fd VI is co-induced with the previously characterized enzymes involved in nitrate assimilation, and they suggest that the novel Fd isoprotein, distinct from the constitutively expressed Fd, might play an important role as an electron carrier from NADPH to nitrite reductase and other Fd-dependent enzymes in root plastids.

The nm23 gene is a potential metastasis suppressor gene originally identified using a murine melanoma cell line. The nm23 proteins are nucleoside diphosphate (NDP) kinases, and there are two isotypes in human and other mammalian cells. Immunohistochemical analysis with anti-rat NDP kinase (NDP kinase alpha, NDP kinase beta) monoclonal antibodies was performed in deparaffinized sections of 56 specimens of oral squamous cell carcinoma. Detection of these NDP kinases in tissue samples was correlated with clinical and histopathological factors. The NDP kinase A (nm23 H1 protein)-positive group was apt to have a lower frequency of neck metastasis than the negative one (p < 0.01). Consequently, it is suggested that NDP kinase A might be a metastasis suppressor factor that may be useful for predicting tumor metastases in patients with oral squamous cell carcinoma.

The mechanism leading to development of oral cancer has not been completely understood. It is currently believed that alternation of a number of genes can result in the development of epidermoid carcinomas. In this investigation, we used a 9,10-dimethyl 1,2-benzanthracene (DMBA)-induced carcinogenesis in a hamster tongue model to investigate the expression of c-myc, c-Ha-ms proteins and epidermal growth factor receptor (EGFr). During the DMBA-carcinogenesis of the tongue, the number of c-myc protein positive cells were increased in epithelial dysplasia and elevated throughout the process of tumorigenesis. The expression of c-Ha-ras protein was detected in normal epithelium. The level of c-Ha-ras protein expression was decreased in the dysplastic stage, and it was almost negative in squamous cell carcinomas. Detection of EGFr overexpression occurred only after 1-4 weeks of DMBA treatment, at a very early stage of tumor development, and increased through carcinogenesis varying individually within the malignant tissues. These results suggest that c-myc protein and c-Ha-ras protein expression may have an important role in malignant transformation, and the overexpression of EGFr can be correlated to very early stages of tumor development in the DMBA-induced in vivo tongue carcinogenesis.

Immunohistochemical analysis of erbB3, as the third member of epidermal growth factor receptor gene family, was performed on 41 cases of oral squamous cell carcinoma, correlating the staining pattern with clinical outcome. High expression of erbB3 protein (ERBB3) was significantly associated with lymph node metastasis (P < 0.05), survival rate (P < 0.05) and mode of invasion (P < 0.01) in this series. These results demonstrated that ERBB3 expression may be helpful in identifying those oral squamous cell carcinomas with higher malignant potential.

Summary A case of gigantic ameloblastoma of the mandible complicating hypoproteinemia is reported. The patient, a 73-year-old male, had refused a surgical procedure on an ameloblastoma for 13 years. By the time the tumor had increased in size and fistulas from it had formed, hypoproteinemia and generalized edema had occurred. The tumor was removed when the serum total protein level had recovered to about 5 g/dl following the administration of a plasma protein preparation. After the operation, hypoproteinemia and edema clearly improved. Hypoproteinemia is thought to be caused by leakage of plasma or occasional bleeding through the oral fistulas of ameloblastoma, and in this patient's case, poor nutrition because of his masticatory and swallowing difficulties.

The root pulp contained two types of parvalbumin-immunoreactive nerve fibres, smooth and varicose. The smooth were relatively thick (mostly 3–5 μm in diameter) and sometimes had large swellings, whereas the varicose consisted of oval varicosities of 1–3 μm in long diameter with very fine strands interconnecting them. Most of the parvalbumin-immunoreactive root fibres were smooth, and the varicose were rare. Upon entering the pulp chamber, both types of fibre splayed out and ran towards the roof of the chamber and the pulp horn. In the subodontoblastic layer of the roof and horn, parvalbuminimmunoreactive fibres showed relatively straight chains of granules; they sent their granular terminals to the odontoblastic layer. Some parvalbumin-immunoreactive granules were observed in the predentine and some even in the dentine. The diameter of axons in the root pulp and the spatial distribution of terminals strongly suggest that the parvalbumin-immunoreactive fibres are mostly myelinated and sensory.

The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound alpha-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (approximately 1,100 residues), that for glucan binding (approximately 240 residues), and that of unknown function (approximately 260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus.