Yasutaka Kubota

Effects of a phorbol ester on acetylcholine‐induced Ca2+ mobilization and contraction in the porcine coronary artery.

1. The effects of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), an activator of protein kinase C, have been investigated on intact and chemically skinned muscle strips of the porcine coronary artery. 2. In the presence or absence of extracellular Ca2+, TPA (0.1‐1 nM) slightly enhanced the amplitude of ACh (10 microM)‐induced contractions but at 100 nM, inhibited the contractions by approximately 50%. 3. ACh (10 microM) reduced the amount of [32P]phosphatidylinositol 4,5‐bisphosphate (PIP2) and increased the amount of [32P]phosphatidic acid (PA) in the presence or absence of Ca2+. TPA (over 1 nM) dose‐dependently inhibited the hydrolysis of PIP2 induced by ACh. 4. ACh (over 0.1 microM) dose‐dependently increased the intensity of fura‐2 fluorescence in dispersed single‐cell suspensions. TPA (over 1 nM) dose‐dependently inhibited the increase of the Ca2+ transient evoked by ACh, but it did not modify the ionomycin‐induced Ca2+ transient or the resting fluorescence. These inhibitory effects of TPA occurred over a similar dose range to that which inhibited ACh‐induced PIP2 break‐down. 5. When the relationship between ACh‐induced contraction amplitude and Ca2+ transient was observed in the presence or absence of 10 nM‐TPA, TPA greatly reduced the Ca2+ transient but did not modify the amplitude of contraction. 6. In saponin‐treated skinned muscle strips, TPA (10 nM) or 1,2‐diolein (50 micrograms/ml) with phosphatidylserine (PS; 50 micrograms/ml) increased the amplitude of contraction evoked by various concentrations of Ca2+ (0.1‐1.0 microM) without any change in the maximum amplitude of the Ca2+‐induced contraction. 7. TPA (10 nM) with PS (50 micrograms/ml) increased the amplitude of contraction evoked by 10 microM‐inositol 1,4,5‐trisphosphate in chemically skinned muscle strips. 8. It is concluded that TPA inhibits the ACh‐induced [Ca2+]i increase by inhibiting the hydrolysis of PIP2, but that it enhances the Ca2+ sensitivity of the contractile proteins. These results indicate that ACh‐induced contractions are controlled by negative feed‐back regulation of PIP2 hydrolysis together with a positive feed‐back regulation of the Ca2+ sensitivity of the contractile proteins. This may depend on the on‐going level of protein kinase C activation.

Interleukin-1α-dependent Regulation of Matrix Metalloproteinase-9 (MMP-9) Secretion and Activation in the Epithelial Cells of Odontogenic Jaw Cysts:

Interleukin-la (IL-1α) and matrix metalloproteinase-9 (MMP-9) are thought to be involved in odontogenic cyst expansion. In this study, we investigated the effects of IL-1α on the secretion and activation of MMP-9 in odontogenic jaw cysts. An active form of MMP-9 was present in odontogenic keratocyst (6 of 8 cases) fluids more frequently than dentigerous cyst (3 of 10 cases) and radicular cyst (3 of 10 cases) fluids, although proMMP-9 was present in all cyst fluids. Odontogenic keratocyst fragments in explant culture secreted a larger amount of IL-1α than dentigerous cyst and radicular cyst fragments in explant culture, and spontaneously secreted both proMMP-9 and an active form of MMP-9. The fragments of dentigerous cysts and radicular cysts secreted a small amount of proMMP-9, but no active form of MMP-9. Exogenously added recombinant human IL-1α (rhIL-la) increased the secretion and activation of proMMP-9 in the fragments of dentigerous cysts and radicular cysts. The epithelial cells isolated from odontogenic keratocysts secreted IL-la and proMMP-9 without stimulation. Under the cultivation on a fibronectin-coated dish, rhIL-la increased the secretion of proMMP-9 from the epithelial cells in a dose-dependent manner. Moreover, rhIL-la induced the secretion of proMMP-3 and plasminogen activator urokinase (u-PA) from the epithelial cells, and converted the secreted proMMP-3 to the active form in the presence of plasminogen. The secreted proMMP-9 was also activated in the presence of rhIL-la and plasminogen. Hence, our results suggest that IL-1α may up-regulate not only proMMP-9 secretion but also proMMP-9 activation by inducing proMMP-3 and u-PA production in the cyst epithelial cells by autocrine/paracrine regulatory mechanisms.

Signaling Pathways Regulating IL-1α-induced COX-2 Expression

Interleukin-1α(IL-1α) stimulates the production of prostaglandin E2 (PGE2) in odontogenic keratocyst fibroblasts. However, the signaling pathways remain obscure. In this study, we investigated IL-1αsignaling pathways that regulate cyclooxygenase-2 (COX-2) expression in odontogenic keratocyst fibroblasts. IL-1αincreased the expression of COX-2 mRNA and protein, and PGE2 secretion in the fibroblasts. IL-1αincreased the phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). PD-98059, SB-203580, SP-600125, and PDTC—which are inhibitors of ERK1/2, p38, JNK, and nuclear factor-κB (NF-κB), respectively—attenuated the IL-1α-induced COX-2 mRNA expression and activated protein kinase C PGE2 secretion. IL-1α(PKC), and PKC inhibitor staurosporine inhibited IL-1α-induced phosphorylation of ERK1/2, p38, and JNK, and decreased IL-1α-induced COX-2 mRNA expression. Thus, in odontogenic keratocyst fibroblasts, IL-1αmay stimulate COX-2 expression both through the PKC-dependent activation of ERK1/2, p38, and JNK signaling pathways, and through the NF-κB cascade.

Effects of Positive Pressure in Odontogenic Keratocysts

Intracystic fluid pressure is thought to be involved in odontogenic cyst growth. In this study, we investigated the effects of positive pressure on the expression of interleukin-1α (IL-1α), matrix metalloproteinases (MMPs), and prostaglandin E2 (PGE2) in odontogenic keratocysts to determine whether this pressure stimulates inflammatory cytokine production and signaling of osteoclastogenic events. Positive pressure enhanced the expression of IL-1α mRNA and protein in odontogenic keratocyst epithelial cells, and increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in a co-culture of odontogenic keratocyst fibroblasts and the epithelial cells. The pressure-induced secretions were inhibited by an interleukin-1 receptor antagonist. Recombinant human interleukin-1α (rhIL-1α) increased the secretion of MMP-1, MMP-2, MMP-3, and PGE2 in the fibroblasts. Furthermore, in the fibroblasts, rhIL-1α enhanced the expression of macrophage colony-stimulating factor (M-CSF) mRNA, and rhIL-1α-induced PGE2 increased the expression of nuclear factor κB ligand (RANKL) mRNA. Thus, positive pressure may play a crucial role in odontogenic keratocyst growth via stimulating the expression of IL-1α in epithelial cells.

Interleukin-1α Enhances Type I Collagen-induced Activation of Matrix Metalloproteinase-2 in Odontogenic Keratocyst Fibroblasts

Interleukin-1alpha (IL-1alpha) is strongly expressed in odontogenic keratocysts. In this study, we investigated the effects of IL-1alpha on the activation of matrix metalloproteinase-2 (MMP-2) in the fibroblasts isolated from odontogenic keratocysts. Odontogenic keratocyst fibroblasts secreted a latent form of MMP-2 (proMMP-2) spontaneously. Type I collagen induced the activation of the proMMP-2, and recombinant human IL-1alpha (rhIL-1alpha) further enhanced the type I collagen-induced activation of proMMP-2 in a dose-dependent manner. The rhIL-1alpha-induced activation of proMMP-2 was inhibited by anti-human IL-1alpha antibody. A reverse-transcription/polymerase chain-reaction (RT-PCR) and Western immunoblotting demonstrated that the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA and protein was increased in the fibroblasts when the cells were cultured on type I collagen, and the expression was further enhanced by rhIL-1alpha. Thus, IL-1alpha may up-regulate proMMP-2 activation by increasing the expression of MT1-MMP in the fibroblasts isolated from odontogenic keratocysts synergistically with type I collagen.

Marsupialisation for keratocystic odontogenic tumours in the mandible: longitudinal image analysis of tumour size using 3D visualised CT scans

The purpose of this study was to determine how keratocystic odontogenic tumours (KCOTs) in the mandible are reduced during marsupialisation, and to predict the best time for secondary enucleation by analysing computed tomography (CT) images. 15 patients with KCOTs were treated with marsupialisation surgery, and 42 series of CT data taken during the marsupialisation process were analysed. CT data were reconstructed in three-dimensional (3D) images. The 3D images were used to measure the diameter and volume, and to analyse the changes that occurred after marsupialisation. Marsupialised KCOTs tended to be reduced equally towards the window in the tumour. The amount of volume reduction per day (V(r)) was reduced in proportion to the volume (V) with the formula V(r)=-0.0029×V. The formula manipulation for V was V=V(0)×e(-0.0029t) (t=duration after marsupialisaton (day)). The volume of marsupialised KCOTs was reduced by half over a 239 day cycle. These results demonstrate that the future shape of marsupialised mandibular KCOTs, under good control, could be predicted with significant accuracy using CT data. This prediction could decrease the prolonged marsupialisation state in patients with KCOTs.

Actions of a phorbol ester on factors regulating contraction in rabbit mesenteric artery.

The effects of 12-o-tetradecanoylphorbol-13-acetate (TPA) on isometric tension, unloaded shortening velocity determined using the slack test (Vmax), and phosphorylation of myosin light chain (MLC20) were investigated in intact and skinned muscle tissues of the rabbit mesenteric artery. In intact muscles exposed to 128 mM K+, the Vmax reached a maximum before the peak of the phasic component of contraction and reduced proportionally with changes in the amplitude of the subsequent tonic-contraction. TPA (0.1 μM) enhanced to a greater extent the amplitude of the tonic than the phasic contraction. During the tonic phase, TPA enhanced the Vmax and amplitude of the contraction more than the phosphorylation of MLC20. In skinned muscles, Ca2+ (0.3−1.0 μM) increased the force, Vmax and MLC20 phosphorylation, in a dose dependent manner. The relation between any two of the above three parameters was always linear. TPA (0.1 μM) with phosphatidylserine (50 μ/ml) time-dependently increased the force and Vmax observed with 0.3–0.5 μM Ca2+. The relation between the force and Vmax shifted in the direction of that observed in the presence of high Ca2+, but in 0.5 μM Ca2+, TPA with phosphatidylserine showed little change in the phosphorylation of MLC20. As estimated from the actions of TPA, protein kinase C may contribute to activation of contractile proteins through MLC20 phosphorylation dependent and other regulatory processes. (Circulation Research 1988;63:893-902)

Eruption prediction of mandibular premolars associated with dentigerous cysts.

OBJECTIVE A tooth with a dentigerous cyst (DC) does not always erupt by marsupialization. The eruption duration and conditions of DC-associated premolars were examined to predict such eruption following marsupialization. STUDY DESIGN The eruption and conditions including depth, root formation, inclination, and eruption space were examined retrospectively in 21 DC-associated mandibular premolars using dental and panoramic radiograms. RESULTS Fifteen of 21 premolars erupted half within 3 months and all 15 erupted completely within 10 months after marsupialization, without orthodontic traction. The age of the patients, tooth depth, and inclination were significantly different between the erupted and non-erupted groups, whereas there was no significant difference in the root formation or the eruption space between the 2 groups. CONCLUSIONS The successful eruption of a DC-associated premolar can be predicted within 3 months after marsupialization. Furthermore, the eruption may be affected by the patients age, tooth depth, and tooth inclination.

Effects of the patient's age and the size of the primary lesion on the speed of shrinkage after marsupialisation of keratocystic odontogenic tumours, dentigerous cysts, and radicular cysts

Marsupialisation, by which a surgical window is created in the cavity of a cystic lesion, has been recommended to avoid the formation of a bony defect in the jaw and a reduction in a patients quality of life. However, information about the factors that affect the reduction in the size of a cyst after marsupialisation is limited. We have studied the effects of the patients age and the size of the primary lesion on the speed of shrinkage after marsupialisation of keratocystic odontogenic tumours (KCOTs), dentigerous cysts, and radicular cysts. The speed of shrinkage (mm(2)/month) was evaluated by measuring the radiolucent area on panoramic radiographs taken before and after marsupialisation for KCOT (n=28), dentigerous cysts (n=26), and radicular cysts (n=18) in the mandibular molar regions. The mean duration of marsupialisation for each type of cyst was 11 (5), 8 (5), and 5 (2) months, respectively. The radiolucent area decreased linearly in the 3 types depending on the time after marsupialisation: r=-0.86 (p<0.01), r=-0.73 (p<0.01), and r=-0.72 (p<0.01), respectively. The relative speed of shrinkage did not correlate with the age of the patients, though it did correlate with the size of the radiolucent area before marsupialisation in KCOT (r=0.69, p<0.01) and radicular cysts (r=0.73, p<0.01). These results suggest that the size of a cyst before marsupialisation may affect the speed of shrinkage in KCOT and radicular cysts, while the age of the patient does not.

Retrospective Evaluation of Treatment Outcome in Japanese Children With Complete Unilateral Cleft Lip and Palate. Part 1: Five-Year-Olds' Index for Dental Arch Relationships

Objective: To evaluate the dental arch relationships of Japanese children with complete unilateral cleft lip and palate (UCLP) and to examine the 5-year-olds’ index for its validity. Design: Retrospective study and comparison with previous reports. Subjects: One hundred thirty-six children with complete UCLP who received primary cheiloplasty and palatoplasty in the Kyushu University Hospital from 1966 to 1999. Materials: Dental models taken from children 53 to 67 months of age and their cephalograms. Methods: Study models were assessed using five scores; 1 = excellent, 2 = good, 3 = fair, 4 = poor, and 5 = very poor, in accordance with the 5-year-olds’ index and also evaluated using Huddart and Bodenhams numerical classification. Dental arch widths, three-dimensional maxillary dental arch form, and lateral cephalograms were traced and measured. The outcome by 5-year-olds’ index was compared with Huddart and Bodenhams numerical classification, dental arch dimensions, and cephalometric measurements. Results: Occlusal outcome evaluated by the 5-year-olds’ index was rated 2.95, which was classified as fair. This index rating showed a significant relationship with numerical classification and dental arch length, but not with dental arch width. The index showed a relationship with mandibular form and position, but not with maxillary position. Conclusion: The occlusal outcome of the cases with UCLP was fair as evaluated using the 5-year-olds’ index. The index evaluates the anteroposterior relationship of maxillary/mandibular dental arches but does not evaluate the collapse of maxillary segments.