Original Article

99 cases were operated while we could not use antibiotics. The author traced X-ray photos on paper and measured areas of the peeled cavities with a planimeter. Results were as follows. 1) 66 cases had increasing stage and the rates were more than 30 %. 2) Cases with good developments showed larger original areas (50〜100cm^2) and smaller increasing rates (less than 30 %). 3) Also their X-ray photos showed coinciding or almost coinciding lines of the apices of lungs and the bases of cavities, but we had to take precautions against suppuration when they showed a horizontal line several days after operation. 4) Most of too high degree of adhesion or thickning of pleura did not show good results. When we found a cord which we must manage with some procedures by pneumolysis we must attend to suppuration too. 5)We ought to resect 4th or 5th rib more than 20 cm and 5th or 4th several cm supplementary. 6) As a method of constriction we commend the INVAGI.NATION method. 7) The author noticed in a considerable number of cases that the areas of cavities increased again after they kept long balanced stages

Atherosclerosis V, Proceeding of the Fifth International Symposium, A.M. Gotto, L.C. Smith, B. Allen, Spring Verlag, 1979(BOOK REVIEW)

Antiviral effect of micafungin on three strains of human rhinoviruses. H1HeLa cells were infected with human rhinovirus type 14 (A), 21 (B), or 71 (C) (100 CCID50) and immediately treated with indicated concentrations of micafungin. Three days after compound treatment antiviral activity was determined by the reduction of cytopathic effect using MTT assay. Cell viability of DMSO-treated cells was set to 0 % and that of uninfected cells was set to 100 %. (TIF 100 kb

Inactivation of human DGAT2 by oxidative stress on cysteine residues

<div><p>Diacylglycerol acyltransferases (DGATs) have a crucial role in the biosynthesis of triacylglycerol (TG), the major storage form of metabolic energy in eukaryotic organisms. Even though DGAT2, one of two distinct DGATs, has a vital role in TG biosynthesis, little is known about the regulation of DGAT2 activity. In this study, we examined the role of cysteine and its oxidation in the enzymatic activity of human DGAT2 <i>in vitro</i>. Human DGAT2 activity was considerably inhibited not only by thiol-modifying reagents (NEM and IA) but also by ROS-related chemicals (H₂O₂ and β-lapachone), while human DGAT1 and GPAT1 were little affected. Particularly, ROS-related chemicals concomitantly induced intermolecular disulfide crosslinking of human DGAT2. Both the oxidative inactivation and disulfide crosslinking were almost completely reversed by the treatment with DTT, a disulfide-reducing agent. These results clearly demonstrated the significant role of ROS-induced intermolecular crosslinking in the inactivation of human DGAT2 and also suggested DGAT2 as a redox-sensitive regulator in TG biosynthesis.</p></div

Multimeric complex of human DGAT2 formed by H₂O₂-induced disulfide crosslinking in human cells.

<p>Huh-7 cells were transfected with plasmid overexpressing human DGAT2 for 47 hours and further incubated with indicated concentrations of H₂O₂ for 1 hour. Cell extracts were harvested in a way described in Materials and Methods section and subjected to Western blot analysis using anti-DGAT2 antibody (A). The amount of monomeric human DGAT2 proteins presented as redDGAT2 in (A) was quantified and the amount of relative redDGAT2 protein was calculated by setting the values from samples treated with PBS to 100% (B). The mean values and standard deviations were determined from three independent experiments. Asterisks indicate non-specific bands.</p

Susceptibility of human DGAT2 activity to cysteine-specific modifying reagents.

<p>(A) Membrane extracts from human DGAT2-overexpressing Sf9 insect cells were treated with indicated concentrations of NEM or IA. Human DGAT2 activity was measured by using the conventional extraction-based <i>in vitro</i> DGAT assay. The relative DGAT2 activity in percentage was calculated by setting the value from DMSO-treated sample to 100%. (B) Selective inhibitory effect of NEM on human DGAT2 activity compared to that on human DGAT1 and GPAT1. Membrane extracts from human DGAT2-, DGAT1-, or GPAT1-overexpressing Sf9 insect cells were treated with indicated concentrations of NEM or DMSO. Human DGAT1, DGAT2, and GPAT1 activity was measured by using the conventional extraction-based <i>in vitro</i> assays which are described in detail in the Materials and Methods section. The relative enzyme activity in percentage was calculated by setting the value from DMSO-treated sample to 100%. The mean values and standard deviations were determined from four independent assays.</p

Inhibitory effect of ROS and ROS generator on human DGAT2 catalytic activity.

<p>Membrane extracts from human DGAT2-overexpressing Sf9 insect cells were treated with indicated concentrations of H₂O₂ (A) or β-lapachone (B) in the presence or absence of 20 mM DTT. Human DGAT2 activity was measured by using the conventional extraction-based <i>in vitro</i> assays which are described in detail in the Materials and Methods section. The activities of membrane extracts treated with PBS (instead of H₂O₂) or DMSO (instead of β-lapachone) in the absence of DTT were defined as 100%. The mean values and standard deviations were determined from four independent experiments.</p

Significant role of cysteines in human DGAT2 activity.

<p>(A) The location of cysteine residues in human DGAT2 was depicted by asterisks. The transmembrane domains (TMD) and the highly conserved domain are indicated by black and grey squares, respectively. Black vertical line indicates the HPHG motif. (B) The amount of newly synthesized TG in HEK293 cells overexpressing flag-tagged wild-type, mutants (C87A, C96A, C99A, C172A, C214A and C312A) with single cysteine to alanine substitution and mutant (C0) human DGAT2 with all cysteines to alanine substitution. Wild-type and mutant human DGAT2 were overexpressed in HEK293 cells for 42 hours and incubated in the presence of [¹⁴C] glycerol for additional 6 hours. Newly synthesized [¹⁴C] TG was extracted and measured by TLC analysis. The relative TG synthesis in percentage was calculated by setting the value from pcDNA3 vector-transfected cells to 100%. The mean values and standard deviations were determined from three independent experiments. (C) The immunoblots of wild-type and mutant human DGAT2. Flag-tagged wild-type and mutant human DGAT2 were overexpressed in HEK293 cells for 48 hours. Cell extracts were harvested and subjected to Western blot analysis using anti-flag antibody. Magoh protein was examined as a loading control. (D) The normalized human DGAT2 activities of wild-type and mutant human DGAT2. Relative human DGAT2 activities were determined by dividing the amount of newly synthesized TG in panel B by the relative protein amount in panel C and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181076#pone.0181076.s002" target="_blank">S2 Fig</a>. The normalized activity of wild-type DGAT2-transfected cells was defined as 100%.</p

Multimeric complex of human DGAT2 formed by ROS-induced intermolecular disulfide crosslinking <i>in vitro</i>.

<p>Membrane extracts from human DGAT2-overexpressing Sf9 insect cells were treated with H₂O₂ (A) or β-lapachone (B) in the presence or absence of 20 mM DTT and subjected to Western blot analysis using anti-DGAT2 antibody. The amount of monomeric human DGAT2 proteins presented as redDGAT2 in (A) and (B) was quantified and the amount of relative redDGAT2 protein was calculated by setting the values from samples treated with PBS (C) or DMSO (D) to 100%. Asterisk indicates a non-specific band.</p

Additional file 2: Figure S2. of Antiviral activity of micafungin against enterovirus 71

Micafungin inhibits the replication of CVB replicon. (A) Vero cells were transfected with in vitro transcribed CVB3 replicon RNAs, instantly treated with indicated concentrations of micafungin for 8 hours and then assayed for firefly luciferase activity. Luciferase activity of DMSO-treated cells was set to 100 %. (B) At the same condition another set of CVB3 replicon-transfected cells were assayed for cell viability by using CellTiter-Glo reagent. Activity of DMSO-treated cells was set to 100 %. (TIF 100 kb

Additional file 1: Figure S1. of Antiviral activity of micafungin against enterovirus 71

Time-dependent luciferase activity of EV71 replicon in Vero cells. Vero cells were transfected with in vitro transcribed EV71-replicon RNAs and then firefly luciferase activity was measured at an interval of two hours. (TIF 80 kb