ペプチド転移酵素Sortase A が触媒するtranspeptidation 反応に対するCa2＋濃度、反応温度、E105点変異、外因性金属イオンの効果に関する研究

Sortase A is a transpetidase from Staphylococcus aureus that recognizes five specific amino acid sequences, LPETG, and binds them to polyglycine on the cell wall surface, immobilizing the target protein on the cell wall surface. Sortase A contains one Ca2＋ as a co-factor in its structure, and Ca2＋ plays an important role in the enzymatic reaction. First, the time course of the enzymatic reaction, the optimal Ca2＋ concentration, and the optimal reaction temperature were investigated using wild-type Sortase A. Next, we focused on E105, an amino acid residue that has been reported to play an important role in Ca2＋ binding. We prepared point mutants in which E105 was replaced with non-coordinating Ala residues and coordinating amino acid residues (Asp, Cys, His) with Lewis base moieties on the side chains and examined the enzymatic reaction in the presence of Ca2＋ and its cognate elements, Mg2＋ and Sr2＋.The result was the clarification of the optimal Ca2＋ concentration and the optimal reaction temperature range for the enzymatic reaction. In the case of Ca2＋ in the wild-type and E105 mutants, the reactivity was WT ≈ E105D＜E105C＜E105A＜E105H, indicating that the enzymatic reaction is affected by the Lewis basicity of the ligand and the steric structure of the side chain. For Sr2＋, the same trend of reactivity as Ca2＋ was observed, but the overall reactivity decreased. However, for Mg2＋, reactivity was significantly decreased in all cases, indicating the significant effect of ionic radius on the enzymatic reaction.論

Treatment of a Patient with Class I Malocclusion and Severe Tooth Crowding Using Invisalign and Fixed Appliances

The Invisalign system is widely used to treat mild to moderate tooth crowding. Recently, Invisalign appliances have been used in orthodontic patients with increasingly complex malocclusions. When using an Invisalign appliance to correct severe tooth crowding, root positions must be carefully controlled during extraction space closure. We report our treatment of a 34-year-old man who presented with a Class I relationship, a midline deviation, severe overjet, and severe tooth crowding in the maxillary and mandibular anterior regions. The treatment plan involved extraction of the maxillary and mandibular first premolars on both sides. We treated this patient with severe anterior tooth crowding using an Invisalign appliance combined with a fixed appliance with power arms and elastics. Treatment by Invisalign as an application of computational calculated result, especially in extraction cases, doctors should have professional skill and experience as an orthodontist

Sip1, a conserved AP-1 accessory protein, is important for Golgi/endosome trafficking in fission yeast.

We had previously identified the mutant allele of apm1(+) that encodes a homolog of the mammalian μ 1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex and demonstrated that the AP-1 complex plays a role in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Here, we isolated a mutant allele of its4(+)/sip1(+), which encodes a conserved AP-1 accessory protein. The its4-1/sip1-i4 mutants and apm1-deletion cells exhibited similar phenotypes, including sensitivity to the calcineurin inhibitor FK506, Cl(-) and valproic acid as well as various defects in Golgi/endosomal trafficking and cytokinesis. Electron micrographs of sip1-i4 mutants revealed vacuole fragmentation and accumulation of abnormal Golgi-like structures and secretory vesicles. Overexpression of Apm1 suppressed defective membrane trafficking in sip1-i4 mutants. The Sip1-green fluorescent protein (GFP) co-localized with Apm1-mCherry at Golgi/endosomes, and Sip1 physically interacted with each subunit of the AP-1 complex. We found that Sip1 was a Golgi/endosomal protein and the sip1-i4 mutation affected AP-1 localization at Golgi/endosomes, thus indicating that Sip1 recruited the AP-1 complex to endosomal membranes by physically interacting with each subunit of this complex. Furthermore, Sip1 is required for the correct localization of Bgs1/Cps1, 1,3-β-D-glucan synthase to polarized growth sites. Consistently, the sip1-i4 mutants displayed a severe sensitivity to micafungin, a potent inhibitor of 1,3-β-D-glucan synthase. Taken together, our findings reveal a role for Sip1 in the regulation of Golgi/endosome trafficking in coordination with the AP-1 complex, and identified Bgs1, required for cell wall synthesis, as the new cargo of AP-1-dependent trafficking

The individual adaptin subunit failed to localize at Golgi/endosomes in <i>sip1-i4</i> mutant cells.

<p>Subcellular localization of Apm1-GFP (A), Aps1-GFP (C), GFP-Apl2 (B), and GFP-Apl4 (D) in wild-type (wt) and <i>sip1-i4</i> mutant cells (<i>sip1-i4</i>). Cells expressing various GFP-tagged adaptin subunits were cultured in YPD medium at 27°C were incubated with the dye FM4-64 for 5 min at 27°C to visualize the Golgi/endosomes. The fluorescence of the FM4-64 was examined under the fluorescence microscope. Arrowheads indicated the localization of GFP-tagged adaptin subunits to the Golgi/endosomes. Bar, 10 µm.</p

Vacuole fusion was defective, but the internalization step of endocytosis was not impaired in <i>sip1-62</i> mutant cells and <i>sip1-i4</i> mutant cells.

<p>(A) Time-course analysis of FM4-64 internalization. Wild-type (wt), <i>sip1-i4</i> mutant (<i>sip1-i4</i>), <i>sip1-62</i> mutant (<i>sip1-62</i>), and Apm1-deletion cells (Δ<i>apm1</i>) were incubated in YPD media with FM4-64 at 27°C for 5 min. Washed cells were observed with a fluorescence microscope (FM4-64) at each time point. (B) Time-course analysis of Lucifer yellow (LY) internalization. Wild-type (wt), <i>sip1-i4</i> mutant (<i>sip1-i4</i>), <i>sip1-62</i> mutant (<i>sip1-62</i>), and Apm1-deletion cells (Δ<i>apm1</i>) were incubated in YPD medium containing Lucifer yellow (5 mg/ml) and were processed as indicated in 5A. Bar, 10 µm.</p

Schizosaccharomyces pombe strains used in this study.

<p>Schizosaccharomyces pombe strains used in this study.</p

The colocalization of Syb1 and Vrg4 with FM4-64 in wild-type and <i>sip1-i4</i> mutant cells.

<p>(A) GFP-fused synaptobrevin failed to localize on the cell surface but partially instead accumulated at the Golgi/endosomes in <i>sip1-i4</i> mutant cells similar to those seen in Apm1-deletion cells (Δ<i>apm1</i>). Wild-type (wt), <i>sip1-i4</i> mutant (<i>sip1-i4</i>), <i>sip1-62</i> mutant (<i>sip1-62</i>), and Apm1-deletion cells (Δ<i>apm1</i>) expressing GFP-Syb1 were cultured in YPD medium at 27°C were incubated with the dye FM4-64 for 5 min at 27°C to visualize the Golgi/endosomes. The GFP-Syb1 localization and the FM4-64 fluorescence were examined under the fluorescence microscope. Arrowheads indicated the dot-like structures of GFP-Syb1 and Golgi/endosomes stained with FM4-64, double arrowheads indicated the cytoplasmic accumulation, and arrows pointed to the concentrated fluorescence at the medial region and cell surface. Bar, 10 µm. (B) Percentages of cells in which Syb1 was localized at the cell surface, in wild-type cells (wt), <i>sip1-i4</i> mutant (<i>sip1-i4</i>), <i>sip1-62</i> mutant (<i>sip1-62</i>), and Apm1-deletion cells (Δ<i>apm1</i>). (C) Quantitative analysis of the number of Syb1 dots that co-localized with FM4-64/cell in wild-type (wt), <i>sip1-i4</i> mutant (<i>sip1-i4</i>), <i>sip1-62</i> mutant (<i>sip1-62</i>) and Apm1-deletion cells (Δ<i>apm1</i>). Cells in B and C were incubated as in A. (D) GFP-fused Vrg4 did not localize to the Golgi/endosomes in <i>sip1-i4</i> mutant cells. Wild-type (wt), <i>sip1-i4</i> mutant (<i>sip1-i4</i>), and <i>sip1-62</i> mutant cells (<i>sip1-62</i>) expressing GFP-Vrg4 were cultured in YPD medium at 27°C were incubated with the dye FM4-64 for 5 min at 27°C to visualize the Golgi/endosomes. The GFP-Vrg4 localization and the FM4-64 fluorescence were examined under the fluorescence microscope. Arrowheads indicated the dot-like structures of GFP-Vrg4 and Golgi/endosomes stained with FM4-64, and arrows pointed to the dot-like structures of GFP-Vrg4 that did not co-localize with FM4-64. Bar, 10 µm. (E) Quantitative analysis of the number of Vrg4 dots that co-localized with FM4-64/cell in wild-type (wt), <i>sip1-i4</i> mutant (<i>sip1-i4</i>), and <i>sip1-62</i> mutant cells (<i>sip1-62</i>). Cells in E were incubated as in D. The data represent means ± standard deviations as the average of three independent experiments with 150 cells in B, C and E.</p