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Background/PurposeNuclear localization of β-catenin is known to associate with malignant transformation of many squamous cell carcinomas. The aim of this study was to compare β-catenin expression in normal human oral epithelium and areca quid chewing associated oral squamous cell carcinomas (OSCCs) and further to explore the potential mechanisms that may lead to induce β-catenin expression.MethodsA total of 40 areca quid chewing-associated OSCCs and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, extracellular signal-regulated protein kinase inhibitor PD98059, glutathione precursor N-acetyl-l-cysteine (NAC), tyrosine kinase inhibitor herbimycin-A, p38 inhibitor SB203580, and phosphatidylinositaol 3-kinase inhibitor LY294002 were added to find the possible regulatory mechanisms.Resultsβ-catenin expression was significantly higher in OSCC specimens than that in normal oral epithelial specimens (p

An ergostane type triterpenoid methylantcinate A (MAA) isolated from the fruiting bodies of Antrodia camphorata inhibited the growth of oral cancer cell lines OEC-M1 and OC-2 in a dose-dependent manner, without cytotoxic to normal oral gingival fibroblast cells. The major mechanism of growth inhibition was apoptosis induction, as shown by flow cytometric analysis of annexin V-FITC and propidium iodide staining, caspase-3 activation and DNA fragmentation. The increased expression of pro-apoptotic Bax, poly-(ADP-ribose) polymerase cleavage, and activated caspase-3 and decreased expression of anti-apoptotic Bcl-2 and Bcl-xL were also observed. These results provide the first evidence that the anti-oral cancer effects of MAA may involve a mechanism through the mitochondrial dependent pathway. Thus, results reported here may offer further impulse to the development of MAA analogues as potential chemotherapeutic targets for oral cancer complications.

Numerous root canals filling materials are available in the field of dentistry, based on various formulas that contain a variety of different and partly mutagenic components, such as epoxy resin sealers, Ca(OH)2-based materials, and zinc oxide-eugenol cements. AH Plus root canal sealer will not release formaldehyde according to the manufacturer, although AH26 does. The purpose of this study was to analyze the leakage of lactate dehydrogenase (LDH) from rat hepatocytes after treatment with AH26 and AH Plus root canal sealers in vitro. Hepatocytes from male Sprague-Dawley rats were used to test the cytotoxicity of AH26 and AH Plus. The root canal sealers were mixed and then dissolved in the dimethyl sulfoxide to final concentrations of 0.01%, 0.04%, and 0.1% (wt/vol), with a dimethyl sulfoxide concentration of0.05%. Dosage-dependent and time-dependent lactate dehydrogenase leakage values were measured and tested by one-way ANOVA. The results showed that both AH26 and AH Plus are toxic to rat hepatocytes. At a low concentration, AH26 had a higher toxicity than AH Plus to rat hepatocytes.

We found that overexpression of RhoGDIβ, a Rho GDP dissociation inhibitor, induced hypertrophic growth and suppressed cell cycle progression in a cultured cardiomyoblast cell line. Knockdown of RhoGDIβ expression by RNA interference blocked hypertrophic growth. We further demonstrated that RhoGDIβ physically interacts with ZAK and is phosphorylated by ZAK in vitro, and this phosphorylation negatively regulates RhoGDIβ functions. Moreover, the ZAK-RhoGDIβ interaction may maintain ZAK in an inactive hypophosphorylated form. These two proteins could negatively regulate one another such that ZAK suppresses RhoGDIβ functions through phosphorylation and RhoGDIβ counteracts the effects of ZAK by physical interaction. Knockdown of ZAK expression in ZAK- and RhoGDIβ-expressing cells by ZAK-specific RNA interference restored the full functions of RhoGDIβ.

The infiltration of eosinophils into tissues during pathologic responses is often associated with extracellular matrix alterations such as fibrosis. Transforming growth factor-beta 1 (TGF-beta 1) is a well-characterized multifunctional cytokine known to exert potent effects on the extracellular matrix. In this report, we showed the production of TGF-beta 1 by human eosinophils from patients with blood eosinophilia. Northern blot analysis using RNA isolated from eosinophils purified from a patient with the idiopathic hypereosinophilic syndrome (HES) detected the 2.5-kb TGF-beta 1 transcript. In situ hybridization and immunohistochemistry of leukocytes from two patients with HES and two patients with blood eosinophilia localized TGF-beta 1 messenger RNA (mRNA) and protein to eosinophils. No other cell type contained TGF-beta 1 mRNA by in situ hybridization, whereas other leukocytes contained detectable TGF-beta 1 protein by immunohistochemistry. Eosinophils from four normal donors contained little or no detectable TGF-beta 1 protein by immunohistochemistry, whereas eosinophils from two of these four normal donors labeled weakly for TGF-beta 1 mRNA by in situ hybridization. These results show that eosinophils in the peripheral blood of patients with blood eosinophilia can express TGF-beta 1, but that eosinophils in the blood of normal donors contained little or no TGF-beta 1.

To date, there has been very little research into the possible effects of endodontic therapy on regeneration of a lost periodontal attachment. The objective of this study was to examine the effects of the endodontic medication, camphorated parachlorophenol (CMCP), on human periodontal ligament cells in vitro. The cytotoxic effects of CMCP were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay and cell proliferation using a [3H]thymidine incorporation assay. CMCP inhibited the human periodontal ligament cells viability and proliferation in a dose-dependent manner (p0.05). These data indicate that the use of CMCP in a root canal could cause periodontium damage. Although this study was conducted in vitro, the findings suggest that it may not be advisable to use CMCP as an interim medication when a periodontal surgical procedure, especially an attempt at regeneration or a new attachment procedure, is being considered in tissues adjacent to the endodontically involved tooth.

Loss-of function mutations in transmembrane inner ear expressed (Tmie/TMIE) gene have been shown to cause deafness in mice and humans (DFNB6). However, the functional roles of TMIE in the cochlea remain unclear. A primary step toward the understanding of the role of TMIE in hearing and its dysfunction is the documentation of its cellular and sub-cellular location within the cochlea, the auditory organ. In this study, we located and determined the cellular expression of Tmie within the rat cochlea using a polyclonal anti-Tmie antibody. The anti-Tmie antibody identified a specific band of 17 kDa in a variety of rat tissues by using Western blot analyses. The expression products of Tmie were also detected in the spiral limbus, spiral ligament, organ of Corti, and stria vascularis by immunohistochemistry analysis and RT-PCR. Our results point out the presence and localization of Tmie products in the cochlea of rat. Knowledge of spatial distribution of Tmie will provide important insight into the mechanisms that lead to deafness due to mutations in the TMIE gene.

Green tea polyphenols are considered beneficial to human health, especially as cancer chemopreventive agents in recent years. Epigallocatechin- 3-gallate (EGCG), the most abundant polyphenol in green tea, has been proven to suppress colonic tumorigenesis in animal and epidemiological studies, whereas its role in the oral carcinogenesis remains to be elucidated.Cytotoxicity, invasion, and migration assays were used to investigate the effects of human oral cancer cell line OC2 cells exposed to EGCG. To look at the precise involvement of EGCG in cancer metastasis, gelatin zymography and casein zymography were performed to evaluate the impacts of EGCG on matrix metalloproteinase (MMP)-2, MMP-9, and urokinase plasminogen activator (uPA) secretion in OC2 cells.EGCG exhibited a dose-dependent inhibitory effect on the invasion and migration of OC2 cells in the absence of cytotoxicity (P0.05). EGCG was also found to decrease the expressions of MMP-2, MMP-9, and uPA in a concentration-dependent manner (P0.05).Taken together, these results suggest that EGCG could inhibit the invasion and migration of human oral cancer cells and that the effects may partially because of the decreased productions of MMP-2, MMP-9, and uPA.

Background: Recently, evidences have shown that tissue type plasminogen activator (t-PA) may play an important role in the pathogenesis of periodontal diseases. However, the mechanisms and signal transduction pathways involved in the production of t-PA in human osteosarcoma cells are not fully understood. Objectives: The purpose of this study was to investigate the caseinolytic activity in human osteosarcoma cell line U2OS cells stimulated with interleukin-1α (IL-1α) or Porphyromonas gingivalis in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Methods: IL-1α and the supernatants of P. gingivalis were used to evaluate the caseinolytic activity in U2OS cells by using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search possible signal transduction pathways, SB203580, U0126, and LY294002 were added to test how they modulated the caseinolytic activity. Results: Casein zymography exhibited a caseinolytic band with a molecular weight of approximately 70 kDa, suggestive of the presence of t-PA. Secretion of t-PA was found to be stimulated with IL-1α and P. gingivalis during a 2-day culture period (p

To investigate the toxicity of fluoride corrosion extracts of stainless steel (SS) and nickel-titanium (NiTi) wires on a human osteosarcoma cell line (U2OS).The SS and NiTi wires were corroded by an electrochemical method with the application of three kinds of electrolytes: 0.2% pH 3.5 acidulated phosphate fluoride (NaF) in artificial saliva, and pH 4 and pH 6.75 artificial saliva solutions. The extracts were analyzed for nickel, chromium, and titanium ions by the atomic absorption method. The extracts were diluted with medium to different concentrations (1, 0.1, and 0.01 microL/mL). The cell survival rate was determined by the ability of test cells to cleave the tetrazolium salt to form a formazan dye.The results were compared using one-way analysis of variance. Differences between the treatment means were analyzed using a Student-Newman-Keuls (SNK) test and were considered significant at P.05. The release of ionic nickel was different in different extract groups (P.05). The SS and NiTi wires in the 0.2% pH 3.5 NaF artificial saliva group caused a dose-dependent decrease in the survival rate (P.05). Survival rates of cells in the groups exposed to extracts of SS and NiTi wires in pH 4 and pH 6.75 artificial saliva solutions showed no statistical differences (P.05).Orthodontic wires in acidulated fluoride saliva solution can cause U2OS cell toxicity.

To evaluate the question of whether or not paclitaxel affects the distribution and metabolism of chemical carcinogens such as 2-aminofluorene (AF) on Sprague-Dawley rats were examined. The AF, acetylated AF and AF metabolites were determined and examined by using high performance liquid chromatography. After having received AF only, AF with paclitaxel at the same time and paclitaxel pretreated for 24 h then treated with AF for 24 h, urine, stool and tissues such as liver, kidneys, stomach, colon, bladder and blood were collected and assayed for AF and its metabolites. Compared to the control group, paclitaxel caused an increase of the metabolites excreted in urine and stool. The major metabolite excreted in urine and stool was 9-OH-AAF. The liver is the major metabolism center and the major residual metabolite of AF in the liver was also 9-OH-AAF.

Objectives: Five CAPE-like compounds, namely caffeicacid phenethyl ester (CAPE), methyl caffeate (MC), ethyl 3-(3,4-dihydroxyphenyl)acrylate (EC), phenethyl dimethyl caffeate (PEDMC) and phenethyl 3-(4bromophenyl)acrylic (BrCAPE) were tested for their anti-HIV replication in vitro and immune modulation effects in vivo. Methods: Short-term cytotoxicity was assessed by Trypan Blue stain and MTT assay. For antiviral assays, M-tropic (strain JRCSF), T-tropic (strain NL-4-3) and dual tropic (strain 89.6) HIV isolates were used in peripheral blood mononuclear cell (PBMC) culture. Results: None of these CAPE-like compounds showed significant cytotoxicity in the treatment of PBMCs. By P24 EIA tests, CAPE, MC and EC significantly inhibited HIV replication in PBMC cells, but PEDMC and BrCAPEshowedonlyslightlyinhibitoryeffects.Theinvivomodulatoryeffectsonsixcytokines[interleukin (IL)-2, IL-4, IL-6, interferon (IFN)-g, granulocyte-macrophage colony-stimulating factor (GM-CSF) and soluble Fas] were analysed. BALB/c mice treated with different doses or not treated with these CAPE-like chemicals showed that cytokines were increased to different extents by the different treatments. However, the concentrations of IL-6 and GM-CSF were not significantly affected by administration of any of these compounds (P > 0.05). Conclusions:Thedifferenteffectsoftreatmentsonanti-HIVreplicationandcytokinemodulationsuggested that these compounds affect virological and immunological response via different mechanisms. The virological and immunological mechanisms and response to these treatments need to be elaborated in further studies in order to derive the structural features of more effective compounds. Since neither death nor pathological change in the mice were observed in this study, these CAPE-like compounds are worth studying further as potential chemotherapy agents for anti-HIV infection and cytokine modulation.

In this study, we investigated and compared the levels of frictional resistance between metal brackets and orthodontic wires after immersion in an acidified phosphate fluoride (APF) agent.Three types of mandibular incisor stainless steel metal brackets with beta-titanium alloy wire (TMA), heat-activated nickel-titanium wire (Ni-Ti), and 2 sizes of stainless steel wires (SSW) were immersed in 0.2% APF and pH 6.75 artificial saliva solutions for 24 hours. The study included 480 bracket-wire specimens. The frictional resistance was measured on an EZ-test machine (Shimadazu, Tokyo, Japan) with a 5-N load cell. An Alastik (Quik-Stik Clear, A-1 Alastik, 3M Unitek, Monrovia, Calif) module ligated to the bracket was attached to the crosshead of the machine and pulled at a speed of 10 mm per minute for a distance of 5 mm. A completely randomized 1-way ANOVA was used to test for significant differences among the 3 bracket/wire specimens after immersion in 0.2% APF and pH 6.75 artificial saliva solutions. This was followed by the Student-Newman-Keuls multiple comparison of means ranking at P.05 to determine differences between the groups.In the APF-immersed group, the static frictional force was greater than the kinetic frictional force. The frictional forces of the orthodontic wires had statistically significant differences (P.05) in this progressive order: TMA, Ni-Ti, and SSW. Similar frictional force results were obtained in the pH 6.75 saliva group (P.05). The frictional force values of the APF group were higher than those of the pH 6.75 saliva group (P.05).This study demonstrates that the frictional forces of orthodontic brackets and wires are influenced by contact with fluoride-containing solutions.

Insulin-like growth factor-1 (IGF-1) is a member of a family of two interacting polypeptide hormone ligands with close homology to proinsulin. IGF-1 can influence mesenchymal cell migration, proliferation, and extracellular matrix deposition, thus implicating it in the progression of fibrotic disorders. Currently, there is limited information about the regulation of IGF-1 expression in areca quid-associated oral submucous fibrosis (OSF). The aim of this study was to compare IGF-1 expression in normal human buccal mucosa and OSF specimens and further explore the potential mechanism that may lead to induce IGF-1 expression. Twenty OSF specimens and 10 normal buccal mucosa were examined by immunohistochemistry. The activity of IGF-1 from cells cultured from OSF and normal buccal mucosa were by using reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Furthermore, the effect of arecoline, the major areca nut alkaloid, was added to explore the potential mechanism that may lead to induce IGF-1 expression. IGF-1 expression was significantly higher in OSF specimens (p0.05) and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. OSF demonstrated significantly higher IGF-1 protein expression than normal buccal mucosa fibroblast (BMF) both in mRNA and protein levels (p0.05). In addition, arecoline was also found to elevate IGF-1 mRNA and protein expression in a dose-dependent manner (p0.05). Taken together, the data presented here demonstrated that IGF-1 expression is significantly upregulated in OSF from areca quid chewers and arecoline may be responsible for the enhanced IGF-1 expression in vivo.

Chingwaysan, a Chinese herbal formula, contains Cimicfugae Rhizoma, Rehmanniae Radixet Rhizoma, Moutan Radicis Cortex, Coptidis Rhizoma and Angelicae Sinensis Radix. This medicine is well-known for its curing power for ulcerated gums, toothaches, cheek boils and bleeding gingiva. However, no reports can be found on its application in the treatment of oral cancers. We are therefore interested in whether Chingwaysan is capable of causing abnormal apoptosis processes, and whether this condition can be rectified through Chingwaysan herb treatment. We used aqueous extract to treat OC2 and TSCCa cells (both are human oral cancer cell lines) with different Chingwaysan concentrations (0, 10, 25, 50, 75 and 100 μl/ml). The MTT (3, (4, 5-dimethyl-thiazol) 2, 5-diphenyl-tetraxolium bromide) reduction assay was employed to quantify the differences in cell activity and viability. DNA ladder formation on agarose electrophoresis was also performed. The bax expression level was monitored using immunoblotting techniques. The patterns of the changes in expression were scanned and analyzed by NIH image 1.56 software. Taken together, drastic morphological changes, reduced cell viability and the presence of inter-nucleosomal DNA fragmentation all indicated that Chingwaysan is capable of inducing apoptosis in OC2 and TSCCa cell lines. Furthermore, the accumulation of wild type bax protein significantly increased in a dose-dependent manner upon treatment with Chingwaysan. In conclusion, Chingwaysan can induce apoptosis via a bax-dependent pathway in cells from these two particular oral cancer cell lines.

Paclitaxel has been found to exhibit cytotoxic and antitumor activity. There is little information regarding the mechanisms of apoptotic-inducing effect of paclitaxel on human osteogenic sarcoma U-2 OS cells. Several key regulatory proteins are involved in the initiation of apoptosis. Caspase-3 plays a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. We examined the effect of paclitaxel on the cell cycle arrest and apoptosis in U-2 OS cells using flow cytometric analysis and Western blotting. We also measured the inhibition of paclitaxel-induced apoptosis and the caspase-3 activity by the broad-spectrum caspase inhibitor z-VAD-fmk on U-2 OS cells. The increased levels of casapse-3 were also confirmed by cDNA microarray. Our observations were: (1) paclitaxel treatment resulted in G2/M-cycle arrest in U-2 OS cells; (2) time and dose dependent apoptosis of U-2 OS cells was induced by paclitaxel; (3) in U-2 OS cells, z-VAD-fmk blocked the paclitaxel-induced apoptosis and caspase-3 activation. These results suggest that paclitaxel-induced G2/M-cycle arrest of the G2/M phase and apoptosis via a caspase-3 pathway in U-2 OS cells.

The purpose of this study was to compare the prevalence and level of Porphyromonas gingivalis (P. gingivalis) and Tannerella forsythensis (T. forsythensis) in subgingival plaque samples from both healthy individuals and periodontal patients in different age groups.A total of 498 subgingival plaque samples were studied. These samples were collected from 407 individuals diagnosed with periodontal disease (210 adult periodontitis [AP], 78 rapidly progressive periodontitis [RPP], and 119 refractory periodontitis [Ref-P] cases) and 91 healthy (H) subjects. P. gingivalis and T. forsythensis were detected by indirect immunofluorescent assay using species-specific polyclonal antisera to P. gingivalis strain (FDC 381) and T. forsythensis strain (FDC 335). The prevalence of P. gingivalis and T. forsythensis was compared by chi-square analysis. Differences in P. gingivalis and T. forsythensis levels among various periodontal status and age groups was determined by one-way analysis of variance and Fisher's multiple comparison tests. The association between the presence of P. gingivalis or T. forsythensis in different periodontal status and age groups was measured using odds ratios.P. gingivalis was found in 85.7% (P0.0001) and T. forsythensis in 60.7% (P = 0.0002) of diseased subjects compared to 23.1% and 39.6%, respectively, in healthy subjects. P. gingivalis, but not T. forsythensis, was detected more frequently in any diseased group than in the H group in every age group (P0.0001). No significant difference was found in the prevalence of P. gingivalis and T. forsythensis among age groups, except T. forsythensis was more prevalent in the age group of 40 to 59 years than in the age group20 years (chi2 = 3.93, P = 0.047) in the H group. The mean level of P. gingivalis and T. forsythensis was significantly higher in diseased groups than in the H group (P0.0001). The odds ratio for P. gingivalis in the AP group (25.0) was greater than any other group for P. gingivalis or T. forsythensis compared to the H group.These data suggested that P. gingivalis is closely associated with the pathogenesis of periodontitis and that it may not be a normal inhabitant of periodontally healthy subjects. T. forsythensis is also important in the pathogenesis of periodontal disease; however, whether it causes periodontal disease or is a secondary invader of periodontal lesions remains to be determined.

Mouth ulcers are one of the most common oral complaints. However, the association between oral ulceration and viruses and cytokines is uncertain. We detected the presence of human papilloma virus (HPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV)-1, HSV-2 and human herpesvirus (HHV)-8 DNA in oral tissues by polymerase chain reaction (PCR) and Southern hybridization techniques, and quantified the serum levels of cytokines including interleukin (IL)-2, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble Fas (sFas) and the Fas ligand (FasL) by enzyme-linked immunosorbent assay (ELISA) for 67 recurrent aphthous ulcer (RAU) patients and 72 normal individuals. Seven patient specimens were excluded from the study due to the negative PCR results for the beta-globin used as the internal control. Among the 32 (53.3%) virus-positive results from 60 patients' samples, 8 (13.3%) HPV, 4 (6.7%) HSV-1, 11 (18.3%) CMV, 9 (15.0%) EBV, and 16 (26.7%) HHV-8 samples proved to be positive. No HSV-2-positive samples were found. The percentage of single-virus infection (56.3%) was significantly greater than that of double-virus co-infection (31.3%) and the percentage of double-virus co-infection was significantly greater than the percentage of triple-virus co-infection (12.5%) (P0.05). In the 72 normal oral-tissue specimens, no viral DNA was detected. The mean serum cytokine level for patients was significantly (P0.05) greater than for controls for most of the separate age groups. The mean serum cytokine concentrations for the patient group demonstrated a diffuse pattern covering a wide range of serum concentrations, a very different result from the compact serum concentration pattern and lower mean serum cytokine concentrations revealed by the normal group. Overall association between viruses and recurrent aphthous ulceration is HHV-8CMVEBVHPVHSV-1, regarding the frequency of prevalence (P0.05).

Background: Bleomycin A5 is an anti-neoplastic glycoprotein antibiotic used for the treatment of various cancers. Previous work has shown that bleomycin A5 exerts its apoptotic effects on tumor cells. This was to study the signal transduction pathways that might exert the apoptotic effects of bleomycin A5 on tumor cells, as well as to examine the possibility of lower dosing of such drug in combinative treatment with other compounds in vitro. Methods: Bleomycin A5 was applied on a human oral epidermoid carcinoma cell line, human oral epidermoid carcinoma (KB), and the apoptotic activity was determined by the presence of DNA fragmentation and 4,6-diamidino-2-phenylindole (DAPI) nuclear staining. The signal transduction pathway was measured through Western blotting and in vitro kinase assay. Results: The apoptotic effect was associated with the sustained activation of c-Jun N-terminal kinases (JNK) and the inhibition of extracellular signal-regulated kinases1 (ERK1) and -2 activities, suggesting that JNK plays a positive role in the death process. ERK1 and -2 might exert a protection pathway from cell death. Here, it was determined that a combination treatment of bleomycin A5 and the MAP kinase-ERK kinase (MEK) inhibitor, PD98059, could lead to enhanced apoptosis. The activities of ERK1 and -2 are required for cell survival signaling using stable cell clones expressing MEK1. Upon bleomycin A5 treatment, cells expressing MEK1 exhibited significant delays in the onset of apoptosis, where the presence of MEK1 inhibitor enhanced cell death. Moreover, the increased activity of ERK1 and -2 coincided with cell survival. The survival signals exerted by MEK1 most likely result from the activation of ERK1 and -2. Conclusions: The apoptosis enhancement through such combinative treatment in vitro has revealed new therapeutic opportunities and elucidated mechanisms contributing to the efficacy of existing anti-cancer treatments.

Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. COX-2 is an inducible enzyme believed to be responsible for prostaglandin synthesis at site of inflammation. Currently, there is limited information on the regulation of COX-2 expression in smoking-associated periodontal disease.The aim of the present study was to investigate the effects of nicotine on the expression of cyclooxygenase-2 (COX-2) mRNA gene and protein in cultured human gingival fibroblasts (HGFs). Furthermore, to elucidate whether induction of COX-2 may be associated with nicotine- induced cytotoxicity, NS-398 (a selective COX-2 inhibitor), was added to test its protective effect.The quantitative reverse-transcriptase polymerase chain reaction and Western blot assays were used to investigate the effects of human HGFs exposed to nicotine. In addition, NS-398 was added to test how it modulated the effects of nicotine.The exposure of quiescent human HGFs to nicotine resulted in the induction of COX-2 mRNA expression. The levels of the COX-2 mRNAs increased about 1.5 and 2.5 fold after exposure to 2.5 and 15 mm nicotine for 2 h (P0.05), respectively. Moreover, the peak of COX-2 mRNA levels induced by nicotine was 10 mm at 2 h incubation period. Investigations of the time dependence of COX-2 mRNA expression in nicotine-treated HGFs revealed a rapid accumulation of the transcript, a signal first detectable at 30 min and diminished to control level after 8 h. In addition, 10 mm nicotine also induced COX-2 protein expression in HGFs. The kinetics of this response showed that COX-2 was detectable at 4 h and diminished nearly to control level after 24 h. NS-398 at non-cytotoxic dose is not able to prevent nicotine-induced cytotoxicity.Taken together, the activation of COX-2 expression by nicotine suggests a potential role for nicotine in the pathogenesis of smoking-associated periodontal disease. In addition, nicotine-induced cytotoxicity is not directly via the induction of COX-2 expression.

Betel quid chewing is a general oral habit in Taiwan, India, southeastern Asian and South Africa with or without the additive of tobacco, alcohol or lime. In this study, the tumor-promoting neoplastic transformation effect of Lime-Piper betel quid (LPB) was examined on JB6 cells. The treatment of LPB at a high dose (1.0 mg/ml) for over 5 days or at lower doses (0.1, 0.5 mg/ml) for over 15 days induced the formation of transformed foci. The transformed cells showed the characteristics of colony formation in soft agar, higher growth rate and multilayer on culture dish. A two-fold induction of the protein levels of c-fos and c-jun proto-oncogenes was observed in the cells from the 50th passage (Cl1/p50, Cl2/p50 and Cl3/p50), suggesting that LPB-transformed cells were oncogenic. In addition, the LPB-transformed cells possessed an elevated level of c-Myc and an increased cell population distributed in the S phase of the cell cycle. These results demonstrated the promotion effect of LPB and indicate that it could be a tumor promoter.

Methyl methacrylate (MMA) is a volatile liquid widely used in the manufacture of acrylic polymers. In modern dentistry, MMA is the mainstream material in denture bases. MMA has been implicated as primary irritant and sensitizer, which can cause allergic eczematous reaction on the oral mucosa as well as skin. To date, there is growing concern that MMA may produce genetic damage by inducing mutation. In this study, colony forming efficiency, DNA synthesis, and cytogenetic assays were performed to investigate the adverse effects of MMA in cultured CHO cells. MMA was found to decrease colony formation in a dose- and time-dependent manner (P

Aberrant and persistent tissue inflammation are believed to play an important role on the occurrence of tissue fibrosis. Cyclooxygenase (COX)-2 is an inducible enzyme responsible for prostaglandin synthesis in certain inflammatory diseases. The purpose of this study was to compare COX-2 expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induce COX-2 expression.Fifteen OSF specimens and six normal buccal mucosa were examined by immunohistochemistry. Primary human buccal mucosa fibroblasts (BMFs) were established and challenged with arecoline analyzed by reverse transcriptase polymerase chain reaction. Furthermore, to elucidate whether induction of COX-2 is associated with cytotoxicity, aspirin (a non-selective inhibitor of COX enzyme) and NS-398 (a selective COX-2 inhibitor), were added to test their protective effects.COX-2 expression was significantly higher in OSF specimens and expressed mainly by epithelial cells, endothelial cells, and cells with fibroblast morphology. In vitro studies indicated that BMFs did not express COX-2 constitutively. However, when the cells were treated with 80 micro g/ml arecoline, COX-2 expression was up-regulated as early as half an hour. This indicates that COX-2 expression is an early cellular response and regulated by arecoline at transcriptional level. In addition, pre-treatment with glutathione (GSH) precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), led to a decrease in induction of COX-2 mRNA by arecoline. GSH synthesis inhibitor, buthionine sulfoximine (BSO), was found to increase arecoline-induced COX-2 mRNA levels. Moreover, both of aspirin and NS-398 at non-cytotoxic doses are not able to prevent arecoline-induced cytotoxicity. This indicates that arecoline cytotoxicity is not directly via the induction of COX-2 expression.Taken together, these results suggest that COX-2 expression is significantly up-regulated in OSF tissues from areca quid chewers and arecoline may among other constituents be responsible for the enhanced COX-2 expression in vivo. The regulation of COX-2 expression induced by arecoline is critically dependent on the cellular GSH concentration.

Cigarette smoking is associated with increased incidence of periodontal disease and poor response to periodontal therapy. Several studies have shown the detrimental effects of nicotine on periodontal tissue. To investigate the molecular toxicological implications of cigarette smoking on periodontal tissue, expression of c-fos early stress response gene was examined in human periodontal ligament fibroblasts (PDLFs) after exposure to nicotine. The exposure of quiescent human PDLFs to nicotine resulted in the induction of c=fos mRNA expression. The levels of the c-fos mRNAs increased about 2.5 and 4.8-fold after exposure to 2.5 mM and 10 mM nicotine for 2 h, respectively. Moreover, the peak of c-fos mRNA levels induced by nicotine was 5 mM at 2-h incubation period. Kinetic investigations of c-fos mRNA expression in nicotine-treated cells revealed a rapid accumulation of the transcript, a significant signal first detectable after 30 min of exposure. This increase was transient and the level of c-fos mRNAs returned rapidly to that of control cells by 8 h. To determine whether thiol levels were important in induction of c-fos by nicotine, we pretreated cells with the glutathione (GSH) precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. Our results demonstrate that OTZ pretreatment decreased in c-fos mRNA level and BSO pretreatment enhanced in c-fos mRNA level after exposure to nicotine. In addition, nicotine significantly depleted intracellular GSH in a dose-dependent manner (P < 0.05). At a concentration of 5 mM and 20 mM, nicotine depleted about 22.2% and 56% of GSH, respectively. Taken together, c-fos gene expression might be one signal transduction pathway linked to the induction of early response genes by cigarette smoking. These results suggest that the nicotine-dependent stress-specific expression of the c-fos gene correlates with cellular thiol levels in human PDLFs.

The effects of intracellular glutathione (GSH) concentration on the toxicity of pingyangmycin in human squamous cell carcinoma cell line were evaluated. By using the GSH synthesis inhibitor D,L-buthionine-S,R-sulfoximine and the precursor of cysteine 2-oxothiazolidine-4-carboxylate (OTZ), intracellular glutathione levels were artificially changed. After exposed to different GSH concentrations cultured tumor cells were treated with pingyangmycin and the resultant mode of cell death was analyzed using morphological and biochemical criteria. It was found that the toxicity of pingyangmycin was obviously increased to cultured tumor cells on lowering GSH levels, with the mode of cell death switching from necrosis to apoptosis. In contract, treatment with OTZ increased GSH level compared with that of control cells, inhibited cell death induced by pingyangmycin via a necrotic rather than apoptotic process. These observations suggest that modulation of GSH levels effects the toxicity of pingyangmycin and that GSH influences the mode of cell death induced by pingyangmycin.

An important requirement for a dentin bonding agent is biologic compatibility; the bonding agent usually remains in close contact with living dental tissues over a long period of time. Information on the genotoxicity/mutagenicity and cacinogenicity potentials of dentin bonding agents is rare. It has been shown that c-fos and c-jun are induced rapidly by a variety of chemical and physical stimuli. Little is known about the induction of cellular signaling events and specific gene expression after cell exposure to dentin bonding agents. Therefore, we used primary human gingival fibroblasts to examine the effect of six dentin bonding agents on the expression of c-fos and c-jun protooncogenes to evaluate the genotoxicity/mutagenicity and cacinogenicity potential of the dentin bonding agents. The levels of mRNA were measured by the quantitative RT-PCR analysis. c-fos and c-jun mRNA expression in dentin bonding agents-treated cells revealed a rapid accumulation of the transcript, a significant signal first was detectable after 1 h of exposure. Persistent induction of c-jun and c-fos protooncogenes by dentine bonding agents may distribute systemically to cause some unexpected adverse effects on human beings. It would be necessary to identify the severely toxic compounds and replace these substances by better biocompatible components. Otherwise, leaching of those genotoxicity/mutagenicity and cacinogenicity components must be minimized or prevented.

AIM: The purpose of this study was to investigate the expression of cyclooxygenase-2 (COX-2) in radicular cysts. METHODOLOGY: Thirty biopsy specimens of radicular cysts were examined using immunohistochemistry. A peroxidase-labelled streptavidin-biotin technique was used for identification of the COX-2. Fisher's exact test (two-tail) was used for statistical analysis of the results. RESULTS: The result demonstrated that COX-2 expression was significantly higher in radicular cysts with higher levels of inflammatory infiltrates. COX-2 stain was detected in the lining epithelium, subepithelial fibroblasts, macrophages and endothelial cells in all specimens. CONCLUSIONS: COX-2 expression is significantly higher in radicular cysts. COX-2 may play an important role in the pathogenesis of radicular cysts.

The association between oral squamous cell carcinoma (OSCC) and human papillomavirus (HPV) 6, 11, 16 and 18 is uncertain. Past reports varied in the methodology and results. We conducted this study using in situ PCR in situ hybridization (ISH) assay which was considered as the most sensitive method for detection of viral DNA. We undertook an epidemiologic survey about the history of betel quid chewing and cigarette smoking, since these habits are common in Taiwan.In situ PCR ISH was performed on the tumor specimens from 29 patients with OSCC and the oral mucosal specimens from 29 patients without OSCC. Their betel quid chewing and cigarette smoking histories were also reviewed.HPV16, HPV18, betel quid chewing and cigarette smoking were statistically significant risk factors in univariate analysis. HPV6 and 11 were not. Multivariate analysis showed that HPV16 infection (adjusted Odds ratio = 11.20) and betel quid chewing (adjusted Odds ratio = 17.06) remained to be independent factors for OSCC.Our results showed that HPV16 and betel quid chewing were two major risk factors for OSCC in Taiwan, indicating that they act through different mechanisms in the pathogenesis of OSCC.

N-Acetylation is recognized as the first step in arylamine metabolism. The enzyme responsible for N-acetylation is called arylamine N-acetyltransferase (NAT),which uses acetyl coenzyme A as the acetyl group donor. Paclitaxel has been shown to exhibit antineoplastic and anticancer activity. In this study, paclitaxel was selected to determine the inhibition of arylamine N-acetyltransferase activity, gene expression (NAT mRNA) and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cell line. Paclitaxel (0.01-l microM) did decrease the level of NAT mRNA in a dose-dependent manner. The results demonstrated that paclitaxel inhibited NAT activity and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cells in a dose-dependent manner. Using standard steady-state kinetic analysis, it was demonstrated that paclitaxel was a possible uncompetitive inhibitor to NAT activity in cytosols based on the decrease in apparent values of K(m) and V(max). This report is the first demonstration that paclitaxel affected human leukemia HL-60 cells NAT activity and DNA-2-aminofluorene adduct formation.

Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in areca quid chewers. It is probably a consequence of disturbances in the hemeostatic equilibrium between synthesis and degradation of extracellular matrix molecules (ECM). To date, there has been little research about the role of tissue inhibitors of metalloproteinase (TIMPs) and matrix metalloproteinases (MMPs) in the pathogenesis of OSF. In the present study, we examined the activity of TIMPs from cells cultured from OSF and normal buccal mucosa. OSF specimens were found to have higher TIMP-1 expression than normal buccal mucosal fibroblasts (BMFs) by Western blots. To verify whether arecoline, a major areca nut alkaloid, could affect TIMP or MMP production by human BMFs, Western blots and gelatine zymography were used. Arecoline was found to elevate TIMP-1 expression at the concentration level under 20 microg/ml in a dose-dependent manner. The amount of TIMP-1 was about 2.7 fold at a concentration level of 10 microg/ml compared with control. From gelatin zymograms, the main gelatinolytic proteinase secreted by the human BMFs was MMP-2, and only minimal amounts of MMP-9 could be detectable from zymogram. In addition, arecoline was found to inhibit MMP-2 secretion and production at the concentration level of 40 microg/ml. The gelatinolytic activity of MMP-2 was about 54% at a concentration level of 80 microg/ml compared with control. Taken together, it was found that arecoline acted not only as an inhibitor on gelatinolytic activity of MMP-2, but also a stimulator for TIMP-1 activity. These synergistic effects may contribute to the ECM components accumulation in the areca quid associated OSF.

The antibacterial effects of various types of widely used endodontic sealers have not been compared systematically on facultative or obligate anaerobic endodontic pathogens. The aim of this study was to evaluate the antimicrobial properties of four commonly used endodontic sealers: two epoxy-resin-based sealers (AH26, AH plus), one zinc-oxide eugenol-based sealer (N2), and one calcium hydroxide-based sealer (Sealapex). The testing microbes were four facultative anaerobic species (Streptococcus mutans, Streptococcus sanguis, Escherichia coli, and Staphylococcus aureus) and four obligate anaerobic species (Porphyromonas gingivalis, Porphyromonas endodontalis, Fusobacterium nucleatum, and Prevotella intermedia). The freshly mixed sealers were placed into the prepared wells of agar plates inoculated with the test microorganisms. After varying periods of incubation (2 days for facultative anaerobic species and 7 days for obligate anaerobic species), the zones of growth inhibition were observed and measured. All the sealers were distinctly different from each other in their antimicrobial activity. The sealers showed different inhibitory effects depending on the types and bacterial strains. N2 containing formaldehyde and eugenol proved to be the most effective against the microorganisms. The extreme antimicrobial potency of this root canal sealer must be weighted against its pronounced tissue toxic effect.

Background Matrix metalloproteinase-11 (MMP-11) is reported to be overexpressed in several cancers and may contribute to tumorigenesis. The current study investigated the association between the clinicopathological characteristics and plasma level of MMP-11 in oral squamous cell carcinoma (OSCC) patients. Methodology and Principal Findings The plasma MMP-11 concentration was determined by ELISA on 330 male OSCC patients. In addition, the metastatic effects of the MMP-11 knockdown on the oral cancer cells were investigated by cell migration assay. Our results showed that the plasma MMP-11 levels were significantly higher in patients with advanced T status (p = 0.001), lymph node metastasis (p = 0.006) and higher TNM stages (p

Objective: The objective of this study was to examine the effects of sodium hypochlorite (NaOCl) and chlorhexidine (CHx) on cultured human periodontal ligament (PDL) cells in vitro. Study Design: The effects of irrigation solutions on human PDL cells were evaluated by propidium iodide fluorescence cytotoxicity assay, protein synthesis assay, and mitochondrial activity. Results: Both NaOCl and CHx were cytotoxic to human PDL cells in a concentration- and contact time–dependent manner. In addition, CHx inhibited protein synthesis in human PDL cells. Although NaOCl displayed cellular cytotoxicity, it showed no protein inhibition in the PDL cells. Furthermore, both NaOCl and CHx exhibited an inhibitory effect on mitochondrial activity on human PDL cells. Conclusions: This study suggests that these irrigation fluids may cause detrimental effects on vital tissues. Its clinical significance, however, needs to be evaluated further because concentration used, exposure time to the agent, and exposure surface area are important factors affecting the resulting effect. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:446-50)

A novel mixed lineage kinase-like protein ZAK, containing a leucine-zipper (L Z ) and a sterile-alpha motif (S A M), was cloned. This cDNA has 2456 bp and encodes a protein of 800 amino acids that contains a kinase catalytic domain, a leucine-zipper and a SAM. The molecular weight of this protein is 91kDa. Northern blot analysis revealed that the expression of this ZAK gene is found in various parts of human tissues. We also found that ZAK proteins might form homodimers or oligomers in mammalian cells. MLKs have been proposed to function as mitogen-activated protein kinase kinase kinase in pathways leading to MAPK cascade. The expression of ZAK in mammalian cells specifically leads to the activation of the JNK/SAPK pathway as well as the activation of transcription factor, NF-κB. Overexpression of the ZAK gene induces the apoptosis of a hepatoma cell line.

The inactivation of tumor suppressor gene (TSG) is important during multistage carcinogenesis. The p53 TSG is frequently mutated in oral squamous cell carcinomas. These mutations can serve as very specific markers for the presence of tumor cells in a background of normal cells. In this study, 10 oral squamous cell carcinoma patients and 27 normal dental students were collected from Chung Shan Medical and Dental College Hospital, Taichung, Taiwan. Extractions of DNA from saliva were obtained. Exon 4 and intron 6 within the p53 gene were amplified with polymerase chain reactions (PCRs) followed by DNA sequence analysis. DNA sequence analysis of PCR products revealed that five of eight (62.5%) tumor saliva samples and five of 27 (18. 52%) healthy saliva samples contained p53 exon 4 codon 63 mutations. These results were significantly different by using Chi-square test (P

Pingyangmycin (PYM; Bleomycin A(5)), an antitumour antibiotic is currently used during anticancer therapy. Previous experiments demonstrated that the therapeutic efficiency of PYM for treatment of malignant tumours is considered to be related to its ability to cause DNA strand breaks in vitro. However, very little is known about the interaction of PYM with the target cells, and it is still unclear how PYM enters the cells. In this study, cell death induced by PYM was studied in a human squamous cell carcinoma cell line (KB cells). In order to determine if cell death occurred by necrosis (reproductive cell death) or apoptosis (programmed cell death), KB cells were exposed to different concentrations of PYM and evaluated by biochemical and morphological criteria. Our results indicate that KB cells displayed an arrest in the G(2)-M phase of the cell cycle and became enlarged and polynucleated before dying at the low concentrations of PYM. In contrast, when cells were exposed to high concentrations of PYM, morphological changes identical to those usually associated with apoptosis were observed as well as internucleosomal digestion of genomic DNA. In conclusion, we demonstrate that PYM is able to induce two distinct modes of cell death depending on the doses of PYM.

Arecoline, a major betel nut alkaloid, has been detected in saliva obtained during betel nut chewing in concentrations up to 140 micrograms/ml, corresponding to 0.9 mM. Arecoline in the millimolar concentration range might participate in the initiation and/or progression of periodontal disease during the long-term effects of betel nut chewing. In this study, cell growth, cell proliferation, assessment of cytoplasmic enzyme lactate dehydrogenase (LDH) and collagen synthesis were used to investigate the effects of human gingival fibroblasts exposed to arecoline levels of 0-200 micrograms/ml. Control culture exhibited a normal monolayer of long spindle-shaped fibroblast morphology. Arecoline-treated human gingival fibroblasts showed a more rounded appearance and detached at the higher concentrations. At concentrations higher than 75 micrograms/ml, many cells had detached from the surface of the petri dish and numerous floating cells could be seen under the inverted microscope. At a concentrations higher than 25 micrograms/ml, arecoline inhibited cell growth, proliferation and collagen synthesis and increased LDH leakage in a dose-dependent manner (P0.05). These results indicate that arecoline is a cytotoxic agent to human gingival fibroblasts. Repeated and long-term exposure to arecoline could impair gingival fibroblast function. Betel quid chewers might be more susceptible to destruction of the periodontium and less responsive to a regeneration procedures during periodontal therapy.

The antitumour antibiotic pingyangmycin (PYM; bleomycin A5) was isolated from many components of bleomycin (BLM) produced by Streptomyces pingyangensisn. PYM has a similar chemical structure to that of BLM but the terminal amine moiety is different. Therefore, it would be of significance to demonstrate the antitumour effect and action mechanism of PYM on cultivated tumour cells. In this study, we used the cell growth curve, plating efficiency, and DNA synthesis inhibition assay to demonstrate the cytotoxicity of PYM on cultured KB cells. In the meantime, the morphological variations of drug-treated cells were also observed. In addition, we used the DNA precipitation assay, a simple and rapid assay, for detecting DNA damage caused by PYM on cultured KB cells for potential genotoxicity. Our results indicate that the effect of PYM significantly inhibits the cell growth, colonyforming ability, and DNA synthesis of KB cells in a dose-dependent manner. Furthermore, when treated with 5 micrograms/ml of PYM for 24 h on cultured KB cells, DNA strand breaks can be induced (P0.05). Therefore, it is considered that the action mechanism of PYM is due to its ability to inhibit the synthesis of DNA and split the DNA chains.

Betel quid chewing has been linked to oral submucous fibrosis and oral cancer. Cytotoxicity and genotoxicity assays were used to investigate the pathobiological effects of arecoline on cultured human buccal fibroblasts. Arecoline increased double-stranded polynucleic acid at the concentration of 0.1 to 10 micrograms/ml in a concentration-dependent manner. At a concentration higher than 50 micrograms/ml, arecoline was cytotoxic to cultured fibroblasts and the cytotoxicity was dose-dependent. No genotoxicity for arecoline was found even at a concentration of 400 micrograms/ml. On the other hand, 600 micrograms/ml glutathione (GSH) and 200 micrograms/ml glycyrrhizin could prevent the arecoline-induced cytotoxicity. These results indicate that arecoline is a cytotoxic agent and no genotoxicity was found to human buccal fibroblasts. Furthermore, increasing consumption of GSH- and glycyrrhizin-rich foods may reduce the oral diseases associated with betel quid chewing.

Methyl methacrylate (MMA) is widely used as a cement in dentistry, orthopaedic surgery and ophthalmology. Studies based on short-term genotoxicity tests have produced conflicting results in the last two decades. In the present study, the effects of MMA on the mutagenicity of 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P) were evaluated with the Salmonella typhimurium TA98 strain in the absence and presence of S9 mix. The direct-acting mutagenicity of 1-NP was markedly decreased by MMA in a dose-dependent manner. However, a low inhibitory effect of MMA on the metabolic-acting mutagenicity of B[a]P was observed. MMA did not show mutagenicity within the concentrations of 4.7-37.6 microM either with or without S9 mix. The inhibitory effect of MMA was not due to its cytotoxicity because very low and/or no cytotoxicity of MMA to S. typhimurium TA98 was observed. To confirm the antimutagenicity of MMA against 1-NP and B[a]P, a 32P-postlabelling method was used to determine whether MMA modified DNA adduct formation produced by both compounds in calf thymus DNA. MMA inhibits the formation of 1-NP- and B[a]P-DNA adducts in a dose-dependent manner. The DNA adduct of 1-NP reduced by MMA was greater than that of B[a]P. Thus, we suggested that MMA was possibly acting as an inhibitor of chemical carcinogenesis.

Using the hamster cheek pouch oral cancer model, we have performed a comprehensive analysis of the cytogenetic changes in hamster oral keratinocytes during 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis. Tumour induction in the hamster cheek pouch required repeated application of the carcinogen for 14 weeks. We have found that this hamster oral cancer model to be suitable for cytogenetic studies. Unlike human oral cancers where chromosome breaks have been shown, this is only infrequently observed in DMBA-treated hamster oral keratinocytes. Of importance is the finding that at the beginning of the second week of DMBA treatment, there is a significant increase of karyotypes demonstrating tetraploid or near-tetraploidy. We propose that the significant increase in hamster oral keratinocytes exhibiting tetraploidy be further evaluated as a marker of premalignancy/malignancy.

The psychotropic effects of a calcium channel blocker (Ca antagonist) were examined in behavioral studies following changes in 45Ca2+ influx in synaptosomal fractions of brain tissues using spontaneously hypertensive rats (SHR). Under a novel circumstance utilizing 85-dB noise, SHR demonstrated hyperactivity and a significant increase in 45Ca2+ uptake into synaptosomal fractions of frontal cortex (FC) and hippocampus. Such hyperactivity may be caused not only be seeking behavior but also by stress-induced anxiety. Such hyperactivity was significantly blocked after 10 days of repeated administration of diazepam (DZP), tandospirone (SM-3997; SM), a 5-HT1A anxiolytic, and nitrendipene (Nit), a Ca antagonist. Moreover, repeated administration of DZP, SM and Nit reduced the maximum binding density of 3H-PN200-110 and reduced the 45Ca2+ uptake in FC of SHR. In hippocampus, midbrain, hypothalamus and striatum, the increased ratio of 45Ca2+ uptake was reduced after repeated administration of Nit or SM. These results suggest that the hyperactivity induced by this novel circumstances was reduced by DZP, SM and Nit and may be attributed to inhibition of voltage-dependent Ca channel activities in FC. In addition, Nit may induce anti-anxiety through the modulation of Ca2+ mobilization in the central nervous system.

The expression of transforming growth factor alpha (TGF-alpha) is consistently associated with the malignant transformation of oral mucosal tissues in both hamster and human. The cheek pouch of the Syrian hamster represents an ideal model to elucidate the role of TGF-alpha in epithelial cancer development. A prerequisite for such investigations at the molecular level is to obtain hamster-specific TGF-alpha molecular probes. Here we report the successful cloning of the hamster TGF-alpha cDNA from a hamster oral cancer cell line (HCPC-1) by the polymerase chain reaction technique using synthetic oligonucleotide primers based on human TGF-alpha cDNA sequence. Analysis of the nucleotide sequence of the newly isolated hamster cDNA encoding the portion of the mature TGF-alpha peptide revealed that it is 92.6% (139/150) homologous to that of the rat and 93.3% (140/150) homologous to that of the human sequences. The predicted hamster TGF-alpha amino acid sequence is 96% (48/50) similar to that of human, while 94% (47/50) similar to that of rat. Using this hamster TGF-alpha cDNA as a probe, molecular hybridization experiments revealed that it detects hamster TGF-alpha mRNA with approximately 40 times and approximately 5 times greater sensitivity than similar probes from human and rat origin respectively. This hamster TGF-alpha cDNA should be of great value as a probe to evaluate the role of TGF-alpha in the normal and pathological processes using the hamster as an experimental model.

Transforming growth factor alpha (TGF-alpha) is a pleuripotential cytokine with diverse biological effects, including the ability to influence the proliferation of normal cells or neoplastic epithelial cells. Eosinophils are a subset of granulocytes that normally enter the peripheral tissues, particularly those beneath gastrointestinal, respiratory, and urogenital epithelium, where they reside in close proximity to the epithelial elements. In this study, we demonstrate that the great majority of eosinophils infiltrating the interstitial tissues adjacent to two colonic adenocarcinomas and two oral squamous cell carcinomas labeled specifically by in situ hybridization with a 35S-riboprobe for human TGF-alpha (hTGF-alpha). No other identifiable leukocytes in these lesions contained detectable hTGF-alpha mRNA. We also examined leukocytes purified from a patient with the idiopathic hypereosinophilic syndrome. 80% of these eosinophils, but none of the patient's neutrophils or mononuclear cells, were positive for hTGF-alpha mRNA by in situ hybridization, and 55% of these eosinophils were positive by immunohistochemistry with a monoclonal antibody directed against the COOH terminus of the mature hTGF-alpha peptide. Finally, the identification of the purified eosinophil-associated transcript as hTGF-alpha was confirmed by polymerase chain reaction product restriction enzyme analysis followed by Southern blot hybridization. In contrast to eosinophils from the patient with hypereosinophilic syndrome, the peripheral blood eosinophils from only two of seven normal donors had detectable TGF-alpha mRNA and none of these eosinophils contained immunohistochemically detectable TGF-alpha product. Taken together, these findings establish that human eosinophils can express TGF-alpha, but suggest that the expression of TGF-alpha by eosinophils may be under microenvironmental regulation. Demonstration of TGF-alpha production by tissue-infiltrating eosinophils and the eosinophils in the hypereosinophilic syndrome identifies a novel mechanism by which eosinophils might contribute to physiological, immunological, and pathological responses.