Hematogenous apoptotic mechanism in salivary glands in chronic periodontitis.

OBJECTIVE: The aim of the study is to investigate the apoptotic mechanism in salivary glands in the rat experimental periodontitis model. DESIGN: A rat periodontitis model was prepared by using a ligature around the second upper molar. In the salivary (parotid and submandibular) glands and blood samples, putative apoptotic factors and pathway molecules were investigated in vivo and in vitro. RESULTS: Four weeks of ligation (chronic periodontitis) demonstrated significant apoptotic atrophy of the salivary gland, but one week of ligation (initial periodontitis) did not. In the blood plasma, tumor necrosis factor-α (TNF-α) was increased in the periodontitis model, but interleukin-1ß and -6 were not. TNF-α receptor type 1, which has an intracellular apoptotic pathway, was expressed in the salivary glands of rats. Western blot analysis of cultured rat primary salivary gland cells demonstrated that TNF-α induced cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 in a dose-dependent manner, indicating apoptosis induction. Additionally, we found increment of circulating lymphocytes in the model. Expression of mRNA and immunoreactive cells for the B lymphocyte marker CD19 were increased in the salivary gland in the model. Western blotting showed that coculture with extracted B cells from the periodontitis model increased cleaved PARP in salivary gland cells. CONCLUSIONS: Chronic periodontitis status leads to an increase in circulating TNF-α and B lymphocyte infiltration, resulting in apoptotic atrophy of the salivary gland as a periodontitis-induced systemic response.

Endocytoscopy for in situ real-time histology of oral mucosal lesions.

This study investigated the utility of endocytoscopy, a novel emerging endoscopic system, for in situ real-time histology of oral mucosal lesions. Endocytoscopy involves the use of a contact light microscopy system with 380-fold magnification. With the development of endoscopic instruments, it has become possible to observe the abnormal microvascular and capillary patterns of tumour cells. The resolution of the endoscopic image is improved in situ, and a more detailed diagnosis is possible. In this study, endocytoscopy along with other diagnostic modalities was used in nine patients. Normal mucous membranes and oral malignant lesions were observed. Endocytoscopy enabled the pathological diagnosis of oral malignancies in situ and the observation of both structural and cytological atypia. In the future, it is expected that pathological diagnoses will be made in situ by direct viewing of living cells. This technique has the potential to allow an 'optical biopsy'.

Hypoxia induced by benign intestinal epithelial cells is associated with cyclooxygenase-2 expression in stromal cells through AP-1-dependent pathway.

Cyclooxygenase-2 (COX-2) plays important roles in tumor development. Especially in the early-stage colorectal tumors, COX-2 expression is often observed in the tumor stroma. However, the mechanism regulating such stromal expression of COX-2 remains unknown. In the present study, we simulated the indirect interaction between epithelial cells and stromal cells in the process of colorectal tumor development using an in vitro co-culture model in which NIH3T3 fibroblasts were co-cultured with 'sparsely' or 'densely' populated intestinal epithelial cells, Intestine-407 as a model of premalignant or benign intestinal epithelial cells, and DLD-1 and Caco-2 as models of malignant epithelial cells. COX-2 expression in NIH3T3 fibroblasts was upregulated when co-cultured with the 'dense' epithelial cells regardless of their character. Interestingly, there was pericellular hypoxia in the vicinity of NIH3T3 fibroblasts when co-cultured with 'dense' epithelial cells, and the recovery of the partial pressure of oxygen level resulted in the reduction of enhanced COX-2 expression only in NIH3T3 fibroblasts co-cultured with 'dense' Intestine-407 cells. Furthermore, COX-2 expression was also reduced by the inhibition of transcription factor AP-1. Thus, pericellular hypoxia of the stromal cells caused by densely populated epithelial cells may be one of the potent COX-2 enhancers before completion of malignant transformation during intestinal tumor development.

Arundic acid (ONO-2506) ameliorates delayed ischemic brain damage by preventing astrocytic overproduction of S100B.

After focal cerebral ischemia, the infarct volume increases rapidly within acute infarct expansion (initial 12 to 24 h) and continues slowly during delayed infarct expansion (25 to 168 h). While acute infarct expansion represents progressive necrosis within the ischemic core, delayed infarct expansion starts as disseminated apoptotic cell death in a narrow rim surrounding the infarct border, which gradually coalesces to form a larger infarct. Discovery of a distinct correlation between reactive astrogliosis along the infarct border and delayed infarct expansion in the rodent ischemia model led us to investigate the possible causal relationship between the two events. Specifically, the calcium binding protein S100B exerts detrimental effects on cell survival through activation of various intracellular signaling pathways, resulting in altered protein expression. Arundic acid [(R)-(-)-2-propyloctanoic acid, ONO-2506] is a novel agent that inhibits S100B synthesis in cultured astrocytes. In the rodent ischemia model, this agent was shown to inhibit both the astrocytic overexpression of S100B and the subsequent activation of signaling pathways in the peri-infarct area. Concurrently, delayed infarct expansion was prevented, and neurologic deficits were promptly ameliorated. The results of subsequent studies suggest that the efficacy of arundic acid is mediated by restoring the activity of astroglial glutamate transporters via enhanced genetic expression.

Inhibitory effects of bifemelane on brain Ca2+ channel subtypes expressed in Xenopus oocytes.

Effects of the cerebroprotective agent bifemelane on voltage-dependent Ca2+ channel currents were evaluated in Xenopus oocytes expressing specific Ca2+-channel subtypes. Extracellular perfusion of bifemelane showed a dose-dependent blocking action on both N-type and Q-type Ca2+ channels, but not on cardiac L-type Ca2+ channels expressed in the oocytes, and the inhibitory action on Q-type current was stronger than that on N-type current. The time course of inhibition by bifemelane was comparatively slow; a 20-min perfusion with 1 microM bifemelane was required to reduce the amplitude of the Q-type current to 80% of the control level. When bifemelane was applied intracellularly, the potency and time-course of inhibition was equivalent to that caused by the perfusion of bifemelane. The bifemelane-induced inhibition was voltage-dependent but not use-dependent in Q-type channels since it was apparent at more depolarized potentials but not influenced by the interval of depolarization. These results suggest that bifemelane inhibits the opening of the specific Ca2+ channels located at nerve terminals to suppress excessive neurotransmitter release from neurons in some pathophysiological conditions such as ischemia.

Inhibition of Ca2+ channel current by mu- and kappa-opioid receptors coexpressed in Xenopus oocytes: desensitization dependence on Ca2+ channel alpha 1 subunits.

1. Desensitization of mu- and kappa-opioid receptor-mediated inhibition of voltage-dependent Ca2+ channels was studied in a Xenopus oocyte translation system. 2. In the oocytes coexpressing kappa-opioid receptors with N- or Q-type Ca2+ channel alpha 1 and beta subunits, the kappa-agonist, U50488H, inhibited both neuronal Ca2+ channel current responses in a pertussis toxin-sensitive manner and the inhibition was reduced by prolonged agonist exposure. 3. More than 10 min was required to halve the inhibition of Q-type channels by the kappa-agonist. However, the half-life for the inhibition of N-type channels was only 6 +/- 1 min. In addition, in the oocytes coexpressing mu-opioid receptors with N-type or Q-type channels, the uncoupling rate of the mu-receptor-mediated inhibition of N-channels was also faster than that of Q-type channels. 4. In the oocytes coexpressing both mu- and kappa-receptors with N-type channels, stimulation of either receptor resulted in a cross-desensitization of the subsequent response to the other agonist. Treatment of oocytes with either H-8 (100 microM), staurosporine (400 nM), okadaic acid (200 nM), phorbol myristate acetate (5 nM) or forskolin (50 microM) plus phosphodiesterase inhibitor did not affect either the desensitization or the agonist-evoked inhibition of Ca2+ channels. 5. These results suggest that the rate of rapid desensitization is dependent on the alpha 1 subtype of the neuronal Ca2+ channel, and that a common phosphorylation-independent mechanism underlies the heterologous desensitization between opioid receptor subtypes.

Cyclic AMP-dependent modulation of N- and Q-type Ca2+ channels expressed in Xenopus oocytes.

Xenopus oocytes were used for investigating the cAMP-dependent modulation of N- and Q-type Ca2+ channels. Treatments to increase intracellular cAMP concentration with forskolin (FK) and 3-isobutyl-1-methylxanthine (IBMX) markedly potentiated Q-type Ca2+ channel current in oocytes coexpressing alpha 1A and beta subunits, and the enhancement was reversed by protein kinase A inhibitors. Moderate enhancement was observed by FK+IBMX in N-type channel current, of which potentiation was equivalent to that of endogenous Ca2+ channel current being activated by exogenously-expressed beta subunits. No potentiation was observed in the oocyte-native Ca2+ channel current. These results suggest that Q-type Ca2+ channels are more susceptible to the protein kinase A-mediated facilitation than N-type channels. A significant role of Ca2+ channel beta subunits for the cAMP-dependent positive modulation was also suggested.

Ca2+ channel inhibition by kappa opioid receptors expressed in Xenopus oocytes.

Functional coupling between kappa opioid receptors and voltage-dependent Ca2+ channels was studied in the Xenopus oocyte translation system, in which specific RNAs encoding rat kappa opioid receptor, rabbit BI-2 alpha 1 subunit, and human beta subunit were co-injected. Perfusion of the oocytes with U50488H inhibited depolarization-evoked Ba2+ current (IBa) in a reversible manner, showing maximal inhibition of 25% at 1 microM (IC50 = 31 nM). The inhibitory effect of U50488H was desensitized by pre-exposure of the oocytes to U50488H and abolished by the kappa opioid antagonist nor-binaltorphimine and by overnight pretreatment with pertussis toxin. Agents affecting the activity of protein kinase A or C did not affect the U50488H-induced inhibition of IBa. These findings suggest that kappa opioid receptors inhibit the activity of neuronal Ca2+ channels via GTP-binding proteins, without the participation of protein kinase A or C.

Base liberation from nucleotides by superoxide and intramolecular enhancement effect of phosphate group.

The reaction of nucleotides with superoxide gave the corresponding nucleobases in good yield. The base-release reaction of nucleotides due to superoxide was examined and compared to that of nucleosides. Hence, the phosphate moiety greatly enhances the yields, in particular those of adenine from adenosine monophosphates. Superoxide in combination with the phosphate moiety has been revealed to be more active than superoxide alone. The phosphate peroxy radical, generated in situ from superoxide and phosphate, seems to be the species responsible for the formation of the free bases.