A study of unfavorable splits in the sagittal ramus osteotomy with a short lingual cut.

In the bilateral sagittal split osteotomy, a short lingual cut is made on the medial side of the ramus; however, in some cases, a true fracture occurs on the buccal side of the ramus. The purpose of this study was to evaluate the relationship between the splitting pattern of the mandible and the form of the mandible, the surgical technique used, and the postoperative occurrence of relapse after 'unfavorable' splits. The investigation examined 143 patients in whom a short lingual cut was attempted. The rate of unfavorable split was 14.7% (42/286). A strong correlation was observed between the reach of the lateral bone cut to the inferior border of the mandible and an unfavorable split. According to multivariate regression, the factors leading to an insufficient lateral bone cut were the degree of inward curvature of the ramus (P=0.001) and the position of the lateral bone cut (P=0.002). There was no significant difference in relapse between cases of unfavorable and normal splits. An unfavorable split does not affect the prognosis of the occlusion, but it is important to confirm the inward curvature of the ramus and set the position of the lateral bone cut adequately to avoid unfavorable splits.

The accuracy of fine needle aspiration cytology in the clinical diagnosis of minor salivary gland tumours.

The objective of this study was to investigate the accuracy of fine needle aspiration cytology (FNAC) and biopsy for the clinical diagnosis of minor salivary gland tumours (MSGTs). This retrospective study of 32 MSGT cases was conducted over a 5-year period. Clinical features including age, sex, and location of the tumour were obtained from the patient clinical records. All cases were also assessed histologically according to the 2017 World Health Organization Classification of Head and Neck Tumours. The results of FNAC and biopsy were correlated with those of histopathology, and their sensitivity, specificity, and diagnostic efficacy were calculated using histopathology as the gold standard. Eighteen malignant MSGTs (56.3%) and 14 benign MSGTs (43.8%) were diagnosed by pathological diagnosis. The most common malignant tumour was mucoepidermoid carcinoma (seven cases, 38.9%). Most benign cases were pleomorphic adenomas (13 cases, 92.9%). FNAC was performed for 23 cases and biopsy for 13 cases. The sensitivity and specificity of FNAC were 66.7% and 91.0%, respectively, while those of biopsy were 90.0% and 100.0%, respectively. Although FNAC is a minimally invasive and cost-effective procedure, it is less accurate than biopsy in the assessment of MSGTs. Repeated FNAC or biopsy should be considered in negative and unsatisfactory FNAC cases.

SIX1 promotes epithelial-mesenchymal transition in colorectal cancer through ZEB1 activation.

Epithelial-mesenchymal transition (EMT) has a major role in cancer progression, as well as normal organ development and human pathology such as organ fibrosis and wound healing. Here, we performed a gene expression array specialized in EMT of colorectal cancer (CRC). From a comprehensive gene expression analysis using epithelial- and mesenchymal-like CRC cell lines, and following the ontology (GO) analysis, SIX1 gene was identified to be an EMT-related gene in CRC. Using SW480 cells stably transfected with a SIX1 expression construct and their control counterparts, we demonstrated that SIX1 overexpression represses CDH1 expression and promotes EMT in CRC. SIX1-induced CDH1 repression and EMT in CRC cells were correlated at least in part with posttranscriptional ZEB1 activation and miR-200-family transcriptional repression. In primary tumors of CRC, in accord with the functional findings, aberrant expression of SIX1 in cancer cells was observed at the disruption of the basement membrane and at the tumor invasive front, where tumor cells underwent EMT in vivo. Taken together, SIX1 overexpression is suggested to occur in carcinogenesis, and contribute to repression of CDH1 expression and promotion of EMT partly through repression of miR-200-family expression and activation of ZEB1 in CRC.

Stabilization of phenotypic plasticity through mesenchymal-specific DNA hypermethylation in cancer cells.

The epithelial-mesenchymal transition (EMT) has a crucial role in normal and disease processes including tumor progression. In this study, we first classified epithelial-like and mesenchymal-like oral squamous cell carcinoma (OSCC) cell lines based on expression profiles of typical EMT-related genes using a panel of 18 OSCC cell lines. Then, we performed methylation-based and expression-based analyses of components of the Wnt signaling pathway, and identified WNT7A and WNT10A as genes silenced by mesenchymal-specific DNA hypermethylation in OSCCs. A significant association was revealed between some clinicopathological findings and the DNA methylation status of WNT7A (normal vs tumor, P=0.007; T1-2 vs T3-4, P=0.040; I-III vs IV, P=0.016) and WNT10A (N0-N1 vs N2-N3, P=0.046) in the advanced stages of OSCC. Moreover, we found that E-cadherin expression in cancer cells may be positively regulated by WNT7A, whose expression is negatively regulated by mesenchymal-specific DNA hypermethylation or ZEB1 in mesenchymal-like OSCC cells. Our findings indicate that epithelial-specific gene silencing through mesenchymal-specific DNA hypermethylation may stabilize the phenotypic plasticity of cancer cells during EMT/MET.

Cytokinesis in Tetrahymena: determination of division plane and organization of contractile ring.

A protein, Tetrahymena p85, is localized to the presumptive division plane before the formation of the contractile ring. p85 directly interacts with Tetrahymena calmodulin (CaM) in a Ca(2+)-dependent manner, and p85 and CaM colocalize in the division furrow. A Ca(2+)/CaM inhibitor N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl (W7) inhibits the direct interaction between p85 and Ca(2+)/CaM. W7 also inhibits the localization of p85 and CaM to the division plane, and the formation of the contractile ring and division furrow. Tetrahymena fimbrin and elongation factor-1a (EF-1alpha), which induce bundling of Tetrahymena F-actin, are also localized to the division furrow during cytokinesis. The Tetrahymena fimbrin has two actin-binding domains, but lacks the EF-hand Ca(2+)-binding motif, suggesting that Tetrahymena fimbrin probably cross-links actin filaments in a Ca(2+)- insensitive manner during cytokinesis. The evidence also indicates that Ca(2+)/CaM inhibits the F-actin-bundling activity of EF-1alpha; and EF-1alpha and CaM colocalize in the division furrow. In this review, we propose that the Ca(2+)/CaM signal and its target protein p85 cooperatively regulate the determination of the division plane, and that a Ca(2+)-insensitive actin-bundling protein, Tetrahymena fimbrin, and a Ca(2+)/CaM-sensitive actin-bundling protein, EF-1alpha, play pivotal roles in regulating the organization of the contractile ring microfilaments.

Tetrahymena elongation factor-1 alpha is localized with calmodulin in the division furrow.

Translation elongation factor 1 alpha (EF-1 alpha) catalyzes the GTP-dependent binding of amino-acyl-tRNA to ribosomes. We previously reported that Tetrahymena EF-1 alpha induced the formation of bundles of rabbit skeletal muscle filamentous actin (F-actin) as well as Tetrahymena F-actin [Kurasawa et al. (1996) Zool. Sci. (Tokyo) 13, 371-375], and that Ca(2+)/calmodulin (CaM) regulated the F-actin-bundling activity of EF-1 alpha [Kurasawa et al. (1996) J. Biochem. 119, 791-798]. In the present study, we investigated the binding between Tetrahymena EF-1 alpha and CaM using a Tetrahymena EF-1 alpha affinity column, and the localization of EF-1 alpha and CaM by indirect immunofluorescence. Only CaM in the Tetrahymena cell extract bound to Tetrahymena EF-1 alpha in a Ca(2+)-dependent manner. In interphase Tetrahymena cells, EF-1 alpha and CaM are colocalized in the crescent structure of the oral apparatus and the apical ring, while in dividing cells, they are colocalized in the division furrow. This is the first report describing the coexistence of EF-1 alpha and CaM in the division furrow, suggesting that EF-1 alpha and CaM are involved in the organization of contractile ring microfilaments during cytokinesis.

Identification of human APC10/Doc1 as a subunit of anaphase promoting complex.

Anaphase-promoting complex or cyclosome (APC) is a ubiquitin ligase which specifically targets mitotic regulatory factors such as Pds1/Cut2 and cyclin B. Identification of the subunits of multiprotein complex APC in several species revealed the highly conserved composition of APC from yeast to human. It has been reported, however, that vertebrate APC is composed of at least eight subunits, APC1 to APC8, while budding yeast APC is constituted of at least 12 components, Apc1 to Apc13. It has not yet been clearly understood whether additional components found in budding yeast, Apc9 to Apc13, are actually composed of mammalian APC. Here we isolated and characterized human APC10/Doc1, and found that APC10/Doc1 binds to APC core subunits throughout the cell cycle. Further, it was found that APC10/Doc1 is localized in centrosomes and mitotic spindles throughout mitosis, while it is also localized in kinetochores from prophase to anaphase and in midbody in telophase and cytokinesis. These results strongly support the notion that human APC10/Doc1 may be one of the APC core subunits rather than the transiently associated regulatory factor.

A new Tetrahymena actin-binding protein is localized in the division furrow.

Using an F-actin affinity column, a 60 kDa fragment of a 71 kDa F-actin-binding protein was partially purified from Tetrahymena pyriformis. After digestion of the 60 kDa fragment with cyanogen bromide, the N-terminal 21-amino acid sequence of one of the resulting peptides was found to show sequence similarity to a region near the actin-binding site (amino acid residues 260-281) of yeast fimbrin. An antibody prepared against a synthesized 21-mer oligopeptide reacted with the 71 kDa proteins in T. pyriformis and T. thermophila cell extracts, suggesting that the 60 kDa fragment was produced from the 71 kDa protein through partial digestion occurring during isolation. The 60 kDa fragment bound to Tetrahymena F-actin as well as to rabbit skeletal muscle F-actin, and induced the bundling of Tetrahymena F-actin. Indirect immunofluorescence revealed colocalization of the 71 kDa protein and actin in the oral apparatus and the deep fiber bundles in T. pyriformis. On the other hand, in T. thermophila, the 71 kDa protein was localized in the oral apparatus and the contractile vacuole pores during the interphase. During cytokinesis, the 71 kDa protein was localized in the division furrow. Therefore, the 71 kDa protein seems to associate with the actin cytoskeleton, and to regulate the actin filament organization during phagocytosis and cytokinesis in Tetrahymena.

Characterization of F-actin bundling activity of Tetrahymena elongation factor 1 alpha investigated with rabbit skeletal muscle actin.

Elongation factor 1 alpha (EF-1 alpha) is an essential factor for protein synthesis in eukaryotes. Here, we demonstrated that Tetrahymena EF-1 alpha induced bundles of rabbit skeletal muscle F-actin as well as Tetrahymena F-actin in vitro, although Tetrahymena and skeletal muscle actins are different in some parts of their primary structures and in the binding abilities to some actin-binding proteins. Co-sedimentation experiments showed that the binding ratio of Tetrahymena EF-1 alpha to skeletal muscle F-actin in the bundles was 1 : 1. Electron microscopic observation showed that alkaline pH or high ionic strength reduced the bundling activity of Tetrahymena EF-1 alpha to some extent, although the EF-1 alpha seemed to be able to induce bundling of the F-actin within the range of physiological condition.

F-actin bundling activity of Tetrahymena elongation factor 1 alpha is regulated by Ca2+/calmodulin.

Translation elongation factor 1 alpha (EF-1 alpha) catalyzes the GTP-dependent binding of amino-acyl-tRNA to the ribosome. Previously, Tetrahymena 14-nm filament-associated protein was identified as EF-1 alpha [Kurasawa et al. (1992) Exp. Cell Res. 203, 251-258]. This and several other studies suggest that EF-1 alpha functions not only in translation but also in regulation of some part of the cytoskeleton. Tetrahymena EF-1 alpha bound to F-actin and induced bundling of F-actin. We investigated the effects of GTP/GDP and Ca2+/calmodulin on F-actin bundling activity of EF-1alpha. The presence of GTP, GDP, or guanylyl-imidodiphosphate (GMP-PNP) slightly decreased the amount of EF-1 alpha which bound to F-actin, but each had virtually no effect on the F-actin bundling activity. The formation of F-actin bundles by EF-1 alpha was Ca(2+)-insensitive. In the absence of Ca2+, calmodulin did not bind to EF-1 alpha and F-actin. On the other hand, in the presence of Ca2+, calmodulin directly bound to EF-1 alpha but did not have any serious influence on EF-1 alpha/F-actin binding. Under the conditions, electron microscopy demonstrated that Ca2+/calmodulin completely inhibited the F-actin bundling by EF-1 alpha. These results indicate that CA2+/calmodulin regulates the F-actin bundling activity of EF-1 alpha without inhibition of the binding between Ef-1 alpha and F-actin.

Polymerization of highly purified Tetrahymena 14-nm filament protein/citrate synthase into filaments and its possible role in regulation of enzymatic activity.

Tetrahymena 14-nm filament protein (49K protein) is a bifunctional protein with roles in the cytoskeleton and as citrate synthase. Previous studies in our laboratory showed that elongation factor 1 alpha (EF-1 alpha) copurifies with the 49K protein upon polymerization and depolymerization of the 49K protein. In this study, the 49K protein was isolated from partially purified 49K protein fraction containing EF-1 alpha. Using the purified 49K protein and/or purified EF-1 alpha, the interaction between 49K protein and EF-1 alpha in filament formation was investigated electronmicroscopically and it was demonstrated that purified 49K protein was capable of forming 14-nm filaments without EF-1 alpha. The 49K protein/citrate synthase has been suggested to form filaments in mitochondria. Here we show that the citrate synthase activity of 49K protein is decreased by polymerization and increased by depolymerization, suggesting a possible modulating mechanism of citrate synthase activity by monomer-polymer conversion in mitochondria in situ.

Identification of Tetrahymena 14-nm filament-associated protein as elongation factor 1 alpha.

Tetrahymena 14-nm filament-forming protein has dual functions as a citrate synthase in mitochondria and as a cytoskeletal protein involved in oral morphogenesis and in pronuclear behavior during conjugation. By immunoblotting using monoclonal and polyclonal antibodies following two-dimensional gel electrophoresis, we demonstrated that the 14-nm filament protein fraction contained two 49-kDa proteins whose isoelectric points were 8.0 and 9.0; a monoclonal antibody (MAb) 26B4 and a polyclonal antibody 49KI reacted only to a pI 8.0 protein, while two other MAbs, 11B6 and 11B8, reacted only to a pI 9.0 protein. From the N-terminal amino acid sequences, the pI 8.0 protein was identified as the previously reported 14-nm filament-forming protein/citrate synthase, but the pI 9.0 protein N-terminal sequence had no similarity with that of the pI 8.0 protein. The pI 9.0 protein is considered to be a 14-nm filament-associated protein since the pI 9.0 protein copurifies with the pI 8.0 protein during two cycles of an assembly and disassembly purification protocol. Cloning and sequencing the pI 9.0 protein gene from a Tetrahymena pyriformis cDNA library, we identified the pI 9.0 protein as elongation factor 1 alpha (EF-1 alpha) based on it sharing 73-76% sequence identity with EF-1 alpha from several species.

Antibodies against Tetrahymena 14-nm filament-forming protein recognize the replication band in Euplotes.

Tetrahymena 14-nm filament-forming protein (49K protein) is a structural protein which is involved in activity of the pronuclei during conjugation (O. Numata, T. Sugai, and Y. Watanabe (1985) Nature (London) 314, 192-194). Using monoclonal and polyclonal antibodies, we here demonstrate the presence of a cross-reactive protein (CRP-49) within the macronuclear replication bands of Euplotes harpa and E. eurystomus which is recognized by anti-49K protein antibodies. Immunoblotting reveals that both monoclonal and polyclonal antibodies cross-react to a protein with an apparent molecular mass of 50 kDa in an E. harpa cell extract and to a protein of 49 kDa in a macronuclear extract of E. eurystomus. The antibodies used in this study have no effect upon in vitro DNA synthesis in the replication band of E. eurystomus.

An unusual clonotypic determinant on a cytotoxic T lymphocyte line is encoded by an immunoglobulin heavy chain variable region gene.

We have detected, in a CTL clone, an mRNA homologous to an immunoglobulin heavy chain variable region (VH) gene segment. A full-length copy of this mRNA was cloned and sequenced, revealing that it is the transcript of an authentic, unrearranged VH gene. The predicted protein product of this expressed VH gene is an approximately 12-kilodalton polypeptide, with secretory signal peptide leader and no membrane-anchoring sequences. Using immunologic reagents generated against a synthetic peptide representing the carboxyl terminus of the VH protein, we detect this protein as a clonotypic cell surface molecule. Strikingly, the anti-peptide reagents also exert effects on CTL function clonotypically. Immunoprecipitation experiments suggest that the VH protein may be associated noncovalently with the consensus, major histocompatibility complex-restricted, antigen-specific T cell receptor alpha- and beta-chains on the cell surface of this CTL. We detect weakly cross-reactive material similar to the VH protein in electrophoretic mobility in other CTL clones and suggest the possibility that small VH-like molecules may constitute a novel class of receptor components with variable determinants involved in the binding of nominal antigen and contributing to overall receptor diversity.

Color reaction of cholesterol with trichloracetic acid and antimony trichloride. On the reaction mechanism.

The color reaction of cholesterol with trichloracetic acid and antimony trichloride was examined to elucidate its reaction mechanism. 3,5-Cholestadiene, 3,3'bis(3,5-cholestadiene), 3,3'bis(2,4-cholestadiene), and cholesteryl trichloroacetate were isolated as the reaction products from the colored reaction mixture of cholesterol, and the first three compounds were found to be responsible for the coloration. It was assumed that cholesterol was dehydrated to 3,5-cholestadiene and 2,4-cholestadiene, which were dimerized to 3,3'-bis(3,5-cholestadiene) and 3,3'-bis(2,4-cholestadiene), respectively, and 3,3'-bis(2,4-cholestadiene) was in part converted to 3,3'-bis(3,5-cholestadiene) in trichloroacetic acid and antimony trichloride. The free radicals were detected in the colored solutions of choelsterol, 3,5-cholestadiene, 3,3'-bis(3,5-cholestadiene), and 3,3'-bis(2,4-cholestadiene), and inferred to be the radical cations of the steroids. The radical cation was postulated to be responsible with respect to the mechanism of the coloration. The relationship between the color reagent and the formation of dimeric steroids was described.