ABSTRACT
OBJECTIVES: The present study aimed to develop a method for measuring the ceiling level of ortho-phthalaldehyde (OPA) exposure and evaluate the ceiling levels of OPA exposure among health care workers who handle disinfectant solutions containing OPA for the disinfection of endoscopes. METHODS: The study consisted of a preliminary survey and main survey. In the preliminary survey, processes involving high-concentration exposure to OPA were identified by video-exposure monitoring (VEM). In the main survey, the ceiling levels of OPA exposure for high-concentration exposure processes identified from the results of the preliminary survey were determined using a measuring method combining sampling using a 2,4-dinitrophenylhydrazine-silica cartridge and analysis by high-performance liquid chromatography tandem mass spectrometry. RESULTS: In the preliminary survey, seven processes involving high-concentration exposure to OPA were identified by VEM. The duration of each process was short, lasting from 20 seconds to a few minutes. In the main survey, the OPA concentrations for the identified high-concentration exposure processes ranged from 1.18 to 4.49 ppb, which markedly exceeded the threshold limit value ceiling (TLV-C) of 0.1 ppb recommended by the American Conference of Governmental Industrial Hygienists. CONCLUSIONS: The method for measuring the ceiling level of OPA exposure was established using VEM and the highly sensitive method of chemical analysis; and we successfully evaluated the ceiling levels of OPA exposure among health care workers engaged in endoscope disinfection. This approach can also be applied to other chemical substances with recommended TLV-Cs, and important information for reducing exposure can thus be obtained.
Subject(s)
Disinfectants/analysis , Endoscopes , Environmental Monitoring/methods , Health Personnel , Occupational Exposure/analysis , o-Phthalaldehyde/analysis , Disinfectants/adverse effects , Equipment Contamination/prevention & control , Humans , Inhalation Exposure , Surveys and Questionnaires , Video Recording , o-Phthalaldehyde/adverse effectsABSTRACT
This chapter deals with the fluorescence detection of SUMOylation and deSUMOylation in semi-intact cultured human cells, the so-called "in situ SUMOylation assay" and the "in situ deSUMOylation assay," respectively. In the in situ SUMOylation assay, the recombinant green-fluorescence protein fused to the SUMO1 (GFP-SUMO1) protein is used to visualize the nuclear rim, nucleolus, and nuclear bodies. These GFP signals represent cellular regions where SUMOylation efficiently takes place. If the recombinant SUMO-specific protease SENP1-catalytic domain is added after in situ SUMOylation, GFP signals can be erased. Therefore, the in situ SUMOylation assay can be used to assess deSUMOylation enzymatic activity.
Subject(s)
Cell Nucleus/enzymology , Cysteine Endopeptidases/metabolism , Green Fluorescent Proteins/metabolism , Protein Processing, Post-Translational , Recombinant Fusion Proteins/metabolism , SUMO-1 Protein/metabolism , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Cell Nucleus/ultrastructure , Cysteine Endopeptidases/genetics , Gene Expression , Green Fluorescent Proteins/genetics , Humans , Hydrolysis , Octoxynol/pharmacology , Protein Binding , Recombinant Fusion Proteins/genetics , SUMO-1 Protein/genetics , SumoylationABSTRACT
Small ubiquitin-related modifier (SUMO) is a highly conserved protein that is covalently attached to target proteins. This posttranslational modification, designated SUMOylation, is a major protein-conjugation-driven strategy designed to regulate structure and function of cellular proteins. SUMOylation consists of an enzymatic cascade involving the E1-activating enzyme and the E2-conjugating enzyme. The SUMO-E1 enzyme consists of two subunits, a heterodimer of activation of Smt3p 1 (Aos1) and ubiquitin activating enzyme 2 (Uba2), which resembles the N- and C-terminal halves of ubiquitin E1 (Uba1). Herein, we describe the rational design of a single polypeptide version of SUMO-E1, a chimera of mouse Aos1 and Uba2 subunits, termed mAU, in which the functional domains appear to be arranged in a fashion similar to Uba1. We also describe the construction of a mAU plasmid for expression in a baculovirus-insect cell system and present an in situ SUMOylation assay using the recombinant mAU. Our results showed that mAU has SUMO-E1 activity, thereby indicating that mAU can be expressed in baculovirus-insect cells and represents a suitable source of SUMO-E1.
Subject(s)
Baculoviridae/genetics , Cloning, Molecular/methods , Protein Engineering/methods , Recombinant Fusion Proteins/genetics , Transduction, Genetic/methods , Ubiquitin-Activating Enzymes/genetics , Animals , Cells, Cultured , Mice , Recombinant Fusion Proteins/biosynthesis , Sf9 Cells , Spodoptera/physiology , Ubiquitin-Activating Enzymes/metabolismABSTRACT
In this study, we cloned the catalytic domain of the Oryzias latipes sentrin/SUMO-specific protease 1 (OlSENP1-CD) gene and produced the recombinant OlSENP1-CD protein in Escherichia coli. Experimental procedures designed to reveal the ability of the recombinant protein to show deSUMOylating activity in vitro should be helpful in future studies of other SENPs and the SUMO pathway.
