Kikuji Yamashita

Cyclosporin A decreases the degradation of type I collagen in rat gingival overgrowth

Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of fibrous gingival overgrowth. The purpose of this study was to investigate the effect of CsA on the type I collagen metabolism in the gingiva of rats fed a powdered diet either containing or lacking CsA. Immunohistochemical analysis revealed that type I collagen was more prevalent in the connective tissue of CsA‐treated gingiva than in those of control rats on days 15, 30, and 55 after the start of feeding. Total RNAs were isolated from mandibular molar gingiva on days 0, 3, 8, 15, 30, and 55. Quantitative analysis of mRNA by reverse transcriptase‐polymerase chain reaction revealed that the CsA‐treated groups showed a gradual decrease in expression of type I collagen and collagenase mRNAs, 0.4% and 18.0% on day 55 compared with those on day 0, respectively. In the control groups, type I collagen and collagenase mRNAs also decreased to 19.7% and 63.0%, respectively, however, both mRNA expressions were significantly lower in the CsA‐treated group than in the controls. An electron microscopic analysis of fibroblasts was performed to count the number of cells with collagen fibrils in the cytoplasm, a marker of phagocytosis of collagen by fibroblasts. The collagen fibrils were detected in 4.7% ± 2.7% and 24.3% ± 13.7% of fibroblasts in the overgrown gingiva treated with CsA rat for 8 days and 30 days, but in 57.0% ± 5.3% and 81.3% ± 9.2% of fibroblasts in the each control group gingiva, respectively. Furthermore, in vitro analysis was performed to measure the phagocytosis of cultured fibroblasts by flow cytometry using collagen‐coated latex beads. Fibroblasts isolated from CsA‐treated gingiva on day 8 and day 30 contained 5.7% ± 0.6% and 9.9% ± 1.5% phagocytic cells, whereas control fibroblasts contained 50.3% ± 5.5% and 33.3% ± 4.9% phagocytic cells, respectively. The inhibition rate of phagocytic activity was similar between in vivo and in vitro assays. These findings suggest that the decrease of the collagen degradation due to the lower phagocytosis and the lower collagenase mRNA expression are closely associated with the increase of type I collagen accumulation in CsA‐treated rat gingiva. J. Cell. Physiol. 182:351–358, 2000.

Ca2+-induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions

Yeast mitochondria have generally been believed not to undergo the permeability transition (PT) by the accumulation of Ca(2+) within the mitochondrial matrix, unlike mammalian mitochondria. However, the reason why the yeast PT is not induced by Ca(2+) has remained obscure. In this study, we examined in detail the effects of Ca(2+) on yeast mitochondria under various conditions. As a result, we discovered that the PT could be induced even in yeast mitochondria by externally added Ca(2+) under optimized experimental conditions. The 2 essential parameters for proper observation of the PT-inducing effects of Ca(2+) were the concentrations of the respiratory substrate and that of inorganic phosphate (Pi) in the incubation medium. The yeast mitochondrial PT induced by Ca(2+) was found to be insensitive to cyclosporin A and suppressed in the presence of a high concentration of Pi. Furthermore, when the PT was induced in yeast mitochondria by Ca(2+), the release of cytochrome c from mitochondria was also observed.

Constitutive expression of thrombospondin 1 in MC3T3‐E1 osteoblastic cells inhibits mineralization

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3‐E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18‐ and 24‐mer antisense oligonucleotides caused concentration‐dependent increases in the number of mineralized nodules, acid‐soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide‐treated MC3T3‐E1 cells, thickened extracellular matrix, well‐developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3‐E1 cells. Furthermore, MC3T3‐E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose‐dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis. J. Cell. Physiol. 209: 322–332, 2006.

Differential Permeabilization Effects of Ca2+ and Valinomycin on the Inner and Outer Mitochondrial Membranes as Revealed by Proteomics Analysis of Proteins Released from Mitochondria

It is well established that cytochrome c is released from mitochondria when the permeability transition (PT) of this organelle is induced by Ca2+. Our previous study showed that valinomycin also caused the release of cytochrome c from mitochondria but without inducing this PT (Shinohara, Y., Almofti, M. R., Yamamoto, T., Ishida, T., Kita, F., Kanzaki, H., Ohnishi, M., Yamashita, K., Shimizu, S., and Terada, H. (2002) Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin. Eur. J. Biochem. 269, 5224–5230). These results indicate that cytochrome c may be released from mitochondria with or without the induction of PT. In the present study, we examined the protein species released from valinomycin- and Ca2+-treated mitochondria by LC-MS/MS analysis. As a result, the proteins located in the intermembrane space were found to be specifically released from valinomycin-treated mitochondria, whereas those in the intermembrane space and in the matrix were released from Ca2+-treated mitochondria. These results were confirmed by Western analysis. Furthermore to examine how the protein release occurred, we examined the correlation between the species of released proteins and those of the abundant proteins in mitochondria. Consequently most of the proteins released from mitochondria treated with either agent were highly expressed proteins in mitochondria, indicating that the release occurred not selectively but in a manner dependent on the concentration of the proteins. Based on these results, the permeabilization effects of Ca2+ and valinomycin on the inner and outer mitochondrial membranes are discussed.

Mastoparan peptide causes mitochondrial permeability transition not by interacting with specific membrane proteins but by interacting with the phospholipid phase

The mastoparan peptide is known as an inducer of the mitochondrial permeability transition. Although mastoparan was suggested to interact with a proteinaceous target in mitochondria to induce this transition, the action sites of mastoparan have not yet been investigated. To clarify whether specific interactions of mastoparan with receptors or enzymes are associated with the induction of this permeability transition, we examined the effects of d‐isomeric peptides, which were synthesized using d‐amino acids assembled in endogenous (inverso mastoparan) and reverse (retro‐inverso mastoparan) orientations. When we added inverso mastoparan to isolated mitochondria, the peptide caused the permeability transition in a partially cyclosporin A‐sensitive manner at lower doses and in a cyclosporin A‐insensitive manner at higher ones. The manners of action and the potencies of inverso mastoparan were close to those of parent mastoparan, indicating that the targets of mastoparan for induction of the permeability transition were neither receptors, nor enzymes in the mitochondria. Retro‐inverso mastoparan also had the same effect on the mitochondria as mastoparan, although the potencies of the effect were weaker. Not only on mitochondria, but also on phospholipid vesicles, mastoparan and inverso mastoparan showed massive permeabilization effects at the same potencies, although retro‐inverso mastoparan showed weaker ones. These results indicate that mastoparan interacted with the phospholipid phase of the mitochondrial membrane (and not with specific proteins) to induce the permeabilization in cyclosporin A‐sensitive and ‐insensitive manners.

Identification of osteopontin in human dental calculus matrix

Osteopontin is a prominent non-collagenous component of bone matrix, although it is expressed in several other tissues. Recently, osteopontin was reported to be involved in urinary stone formation and atherosclerotic lesions of the aorta, suggesting that it may be a key protein associated with these types of pathological mineralization. In this study, whether or not human dental calculus contains osteopontin was investigated by immunoblotting and immunohistochemical analyses. After extraction of calculus proteins with EDTA and separation of the proteins by electrophoresis, immunoblotting analysis revealed the presence of osteopontin. Two forms of osteopontin appeared at 61 and 68 kDa on 10% polyacrylamide gel and the proteins were digested with thrombin, a highly specific protease. Moreover, immunohistochemical analysis revealed that osteopontin was localized in dental calculus adherent to tooth roots. These findings indicate that osteopontin is, in fact, present in human dental calculus and may be involved in calculus formation as the stone matrix.

Gross anatomical study of the human palatopharyngeus muscle throughout its entire course from origin to insertion.

The palatopharyngeus (PP) extends throughout the entire length of the pharynx and probably plays an important role in deglutition, but its spatial distribution remains undefined in some respects. This study was designed to clarify the exact distribution of the PP indispensable for understanding its functions. Using 50 cadavers, the PP and its neighboring muscles were bilaterally exposed in both surfaces of the pharynx. The PP was composed of two divisions: longitudinal and transverse. It is already known that the longitudinal PP is divided into two fasciculi sandwiching the levator veli palatini (LVP) immediately after originating from the palatine aponeurosis. However, we newly discovered a fasciculus originating from the uvula, and further regarded the salpingopharyngeus as another fasciculus of origin. The four fasciculi united to descend through the palatopharyngeal arch and inserted into the thyroid cartilage and beneath the mucosa of the hypopharynx. The transverse PP occupied a location transitional between the PP and superior constrictor (SC), where it originated from the palatine aponeurosis and passed dorsally to encircle the pharyngeal isthmus and reach the pharyngeal raphe. Although whether it belongs to the PP or SC has remained controversial, we regarded it as a portion of the PP from the evolutionary perspective and proposed anatomical criteria for differentiating it from the SC. The wide distribution of the PP suggests that it acts not only to elevate the pharynx or depress the soft palate, but also as a nasopharyngeal sphincter when closing the pharyngeal isthmus. Clin. Anat. 25:314–323, 2012.

cDNA cloning of bovine thrombospondin 1 and its expression in odontoblasts and predentin

The extracellular matrix protein thrombospondin 1 (TSP1) was cloned from odontoblasts of bovine mandibular teeth which participate in dentinogenesis. The 5289 bp cDNA contains a complete open reading frame of 1170 amino acids. Bovine TSP1 has high homologies to its human and mouse counterparts. In immunohistochemical analyses of bovine anterior teeth with anti-TSP1 monoclonal antibody, TSP1 was only detectable at the position of predentin, located between dentin and unmineralized dental pulp. Northern blot analysis showed high levels of two sizes of TSP1 mRNAs in odontoblasts but not dental pulp and gingiva. Previously we found that osteotropic factors such as calcitriol and TGF-beta induce TSP1 at the transcriptional level in clonal rat dental pulp cells. These results suggest a role of TSP1 in dentinogenesis and/or maintenance of dentin and dental pulp.

Non-thermal Effects of Far-Infrared Ray(FIR) on Human Hepatocellular Carcinoma Cells HepG2 and their Tumors

Background: We developed a cell culture CO2 incubatorand a mice rack that can continuously irradiate cells ormurine with FIR. Our goal is to make clear the non-thermaleffect of FIR on HepG2 with these instrumentsmorphologically. Methods: By using them, in vitro , we examined theproliferation of cultured HepG2 cells with hematocytometer,BrdU assay, WST-1 assay, HE staining, Toluidine bluestaining and microarray studies. And in vivo, we measuredthe tumors, observed the sections by IHC, DAPI stainingwith light microscopes and performed microarray studies. Results: Proliferation of HepG2 cells were suppressed(e.g., cell count declined by 34% after 10 days of FIRirradiation), tumor volumes reduced by 86% after 30 daysof FIR irradiation, mRNA of Vascular Endothelial GrowthFactor (VEGF) decreased by 48%, vascular area in crosssections from the tumors decreased 60% compared withthe control. More frequent properties in apoptosis wereobserved by TUNEL and DAPI staining in FIR-treatedgroups. Body weight of mice increased compared with thecontrol. Oxydation and Reduction (Redox) reactions byH+ (proton and electron)/O2- (a kind of Reactive OxygenSpecies (ROS)) were induced by FIR. Conclusions: These results clarified that FIR inhibitedthe proliferation of HepG2 at non-thermal circumstances(at 25±0.5, 37±0.5°C). FIR will serve as a tool againstdiseases induced by HepG2.

Distinct behaviors of adenylate kinase and cytochrome c observed following induction of mitochondrial permeability transition by Ca2+ in the absence of respiratory substrate

For induction of the mitochondrial permeability transition (PT) by Ca2+, the addition of a respiratory substrate such as succinate is required. However, earlier studies indicated the possible induction of the mitochondrial PT by Ca2+ in the absence of a respiratory substrate (Hunter, D.R., and Haworth, R.A. (1979) Arch. Biochem. Biophys. 195, 453–459). In the present study, we obtained clear evidence showing that the mitochondrial PT could be induced by Ca2+ even in the absence of respiratory substrate. We next examined the protein release from mitochondria that accompanied the induction of PT in the absence of a respiratory substrate. Interestingly, distinct from the ordinary mitochondrial PT induced by Ca2+ in the presence of a respiratory substrate, which is associated with the release of mitochondrial cytochome c and adenylate kinase, the mitochondrial PT occurring in the absence of a respiratory substrate was associated with release of mitochondrial adenylate kinase but not with that of mitochondrial cytochrome c. This experimental system should be quite useful for understanding the mechanisms of protein release from mitochondria.