Miki Okayama

Torsional properties and microstructures of miniscrew implants

INTRODUCTION Titanium miniscrew implants are popular in orthodontics, but there is little information about their torsional performance. METHODS Four brands of miniscrew implants (A-D) with 1.6-mm diameters were compared, with miniscrew A implants also having diameters of 1.2 to 2.0 mm. Nominal compositions of the implants were determined by x-ray fluorescence (n = 8). The miniscrews were loaded to failure in torsion, and the mean moment and twist angle were determined for each group (n = 8). Data were compared by ANOVA and the Tukey multiple range tests. Micro x-ray diffraction (n = 3) was used to identify phases in the implants, and the phases were also examined in etched cross-sections with a scanning electron microscope. RESULTS Miniscrew A and C implants were pure titanium, whereas miniscrew B and D implants contained small amounts of vanadium, aluminum, iron, and manganese. Only alpha-titanium peaks were detected for all implants by micro x-ray diffraction, but beta titanium was observed in the microstructures of miniscrew B and D implants, which had significantly higher torsional moments at failure. CONCLUSIONS Addition of small amounts of other elements to titanium yielded significantly improved torsional performance for miniscrew implants. Research to develop optimum compositions for mechanical properties and biocompatibility is needed.

VAMP4 is required to maintain the ribbon structure of the Golgi apparatus

The Golgi apparatus forms a twisted ribbon-like network in the juxtanuclear region of vertebrate cells. Vesicle-associated membrane protein 4 (VAMP4), a v-SNARE protein expressed exclusively in the vertebrate trans-Golgi network (TGN), plays a role in retrograde trafficking from the early endosome to the TGN, although its precise function within the Golgi apparatus remains unclear. To determine whether VAMP4 plays a functional role in maintaining the structure of the Golgi apparatus, we depleted VAMP4 gene expression using RNA interference technology. Depletion of VAMP4 from HeLa cells led to fragmentation of the Golgi ribbon. These fragments were not uniformly distributed throughout the cytoplasm, but remained in the juxtanuclear area. Electron microscopy and immunohistochemistry showed that in the absence of VAMP4, the length of the Golgi stack was shortened, but Golgi stacking was normal. Anterograde trafficking was not impaired in VAMP4-depleted cells, which contained intact microtubule arrays. Depletion of the cognate SNARE partners of VAMP4, syntaxin 6, syntaxin 16, and Vti1a also disrupted the Golgi ribbon structure. Our findings suggested that the maintenance of Golgi ribbon structure requires normal retrograde trafficking from the early endosome to the TGN, which is likely to be mediated by the formation of VAMP4-containing SNARE complexes.The Golgi apparatus forms a twisted ribbon-like network in the juxtanuclear region of vertebrate cells. Vesicle-associated membrane protein 4 (VAMP4), a v-SNARE protein expressed exclusively in the vertebrate trans-Golgi network (TGN), plays a role in retrograde trafficking from the early endosome to the TGN, although its precise function within the Golgi apparatus remains unclear. To determine whether VAMP4 plays a functional role in maintaining the structure of the Golgi apparatus, we depleted VAMP4 gene expression using RNA interference technology. Depletion of VAMP4 from HeLa cells led to fragmentation of the Golgi ribbon. These fragments were not uniformly distributed throughout the cytoplasm, but remained in the juxtanuclear area. Electron microscopy and immunohistochemistry showed that in the absence of VAMP4, the length of the Golgi stack was shortened, but Golgi stacking was normal. Anterograde trafficking was not impaired in VAMP4-depleted cells, which contained intact microtubule arrays. Depletion of the cognate SNARE partners of VAMP4, syntaxin 6, syntaxin 16, and Vti1a also disrupted the Golgi ribbon structure. Our findings suggested that the maintenance of Golgi ribbon structure requires normal retrograde trafficking from the early endosome to the TGN, which is likely to be mediated by the formation of VAMP4-containing SNARE complexes.

SNAP-23 is not essential for constitutive exocytosis in HeLa cells.

We applied the small interfering RNA (siRNA) technique and over‐expression of a dominant‐negative mutant to evaluate the role of SNAP‐23, a non‐neuronal isoform of SNAP‐25, in constitutive exocytosis from HeLa cells. Although the protein level of SNAP‐23 was reduced to less than 10% of the control value by siRNA directed against SNAP‐23, exocytosis of SEAP (secreted alkaline phosphatase) was normal. Double knockdown of SNAP‐23 and syntaxin‐4 also failed to inhibit the secretion. Furthermore, over‐expression of δC8‐SNAP‐23, a dominant‐negative mutant of SNAP‐23, did not abrogate SEAP secretion. These results suggest that SNAP‐23 is not essential for constitutive exocytosis of SEAP.

A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation

DNA methylation is an important epigenetic modification that leads to a wide variety of biological functions, including transcription, growth and development, and diseases associated with altered gene expression such as cancers. However, tools to insert site-specific methylation into DNA for analyzing epigenetic functions are limited. Here we describe a novel polymerase chain reaction (PCR)-based approach to provide site-specific DNA methylation at any site, including CpG or CpNpG islands. This method is simple and versatile, and it consists of four steps to construct the DNA methylation vector: (I) design and synthesis of methylated primers, (II) PCR amplification, (III) isolation of single-stranded DNA, and (IV) annealing and ligation of isolated single-stranded DNAs. First we produced and validated a linear green fluorescence protein (GFP) vector by this method. Next we applied this method to introduce methyl groups into the promoter of the cyclooxygenase-2 (COX-2) gene and found that site-specific DNA methylation at the CRE element significantly altered COX-2 gene expression. These results demonstrate that this PCR-based approach is useful for the analysis of biological functions that depend on DNA methylation.

Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells

AIM The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. METHODS Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. RESULTS Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. CONCLUSION Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement.

Isoproterenol stimulates transient SNAP23–VAMP2 interaction in rat parotid glands

Snap23 physically interacts with Vamp8, Syn‐4 and Vamp2 by anti bait coimmunoprecipitation (View interaction: 1, 2)

SNARE proteins are not excessive for the formation of post-Golgi SNARE complexes in HeLa cells

To evaluate the role of SNARE proteins in the constitutive exocytosis, we knocked down syntaxin 3, 4, 5, 6, 7, and VAMP3, 5, 7, 8 with their siRNAs, and determined the cell-to-medium ratio of CLuc, a secreted luciferase of Cypridina noctiluca. Although the protein level of SNAREs in HeLa cells was markedly reduced by the siRNA treatment, the cell/medium ratio was scarcely increased by any siRNAs except for syntaxin 5. The accumulation of GFP-tagged human growth hormone was also visible only by the knockdown of syntaxin 5. To examine whether the residual amount of SNAREs are sufficient for maintaining normal constitutive exocytosis, we estimated the effect of siRNAs on the level of post-Golgi SNARE complexes containing syntaxin 4, SNAP23, and VAMP3 or VAMP8. The amount of SNARE complexes was robustly decreased by siRNAs and was well correlated with the residual amount of SNAREs in the lysates, suggesting that SNAREs are unnecessarily excessive for the formation of post-Golgi SNARE complexes in HeLa cells.

Cementocyte cell death occurs in rat cellular cementum during orthodontic tooth movement

OBJECTIVE To clarify the mechanism of root resorption during orthodontic treatment, we examined cementocyte cell death and root resorption in the cellular cementum on the pressure side during experimental tooth movement. MATERIALS AND METHODS Using 8-week-old male Wistar rats, the right first molar was pushed mesiobuccally with a force of 40 g by a Ni-Ti alloy wire while the contralateral first molar was used as a control. Localization and number of cleaved caspase-3-positive and single-stranded DNA (ssDNA) - positive cells were evaluated using dual-label immunohistochemistry with anticleaved caspase-3 and anti-ssDNA antibodies. In addition, tartrate-resistant acid phosphatase (TRAP)-positive cells in the cellular cementum were evaluated using TRAP histochemical staining. RESULTS Caspase-3- and ssDNA-positive cells appeared at 12 hours, but were restricted to the compressed periodontal ligament (PDL) and not the cellular cementum. Cleaved caspase-3-positive cementocytes were observed in the cellular cementum adjacent to the compressed PDL on day 1. From days 2 to 4, the number of caspase-3- and ssDNA-positive cementocytes increased. TRAP-positive cells appeared on the cellular cementum at the periphery of the hyalinized tissue on day 7, and resorption progressed into the broad surface of the cementum by day 14. CONCLUSION Cementocytes adjacent to the hyalinized tissue underwent apoptotic cell death during orthodontic tooth movement, which might have been associated with subsequent root resorption.