Publications by authors named "Shinobu Yamamoto"

16 Publications

  • Page 1 of 1

Simple and reliable method to simultaneously determine urinary 1- and 2-naphthol using in situ derivatization and gas chromatography-mass spectrometry for biological monitoring of naphthalene exposure in occupational health practice.

J Occup Health 2020 Jan;62(1):e12144

Osaka Occupational Health Service Center, Japan Industrial Safety and Health Association, Osaka, Japan.

Objectives: The aim of this study was to develop and validate a simple and reliable gas chromatography-mass spectrometry (GC-MS) method to simultaneously determine urinary 1-naphthol (1-NAP) and 2-naphthol (2-NAP) for biological monitoring of occupational exposure to naphthalene.

Methods: NAPs were derivatized in situ with acetic anhydride after enzymatic hydrolysis, extracted with n-hexane, and analyzed using GC-MS. Validation of the proposed method was conducted in accordance with US Food and Drug Administration guidance. A final validation was performed by analyzing a ClinChek -Control for phenolic compounds.

Results: The linearity of calibration curves was indicated by a high correlation coefficient (>0.999) in the concentration range 1-100 μg/L for each NAP. The limits of detection and quantification for each NAP were 0.30 and 1.00 μg/L, respectively. The recovery was 90.8%-98.1%. The intraday and interday accuracies, expressed as the deviation from the nominal value, were 92.2%-99.9% and 93.4%-99.9%, respectively. The intraday and interday precision, expressed as the relative standard deviation, was 0.3%-3.9% and 0.4%-4.1%, respectively. The ClinChek values obtained using our method were sufficiently accurate.

Conclusions: The proposed method is simple, reliable, and appropriate for routine analyses, and is useful for biological monitoring of naphthalene exposure in occupational health practice.
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http://dx.doi.org/10.1002/1348-9585.12144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382817PMC
January 2020

Evaluation of the ceiling levels of ortho-phthalaldehyde exposure among health care workers engaged in endoscope disinfection: A new methodology using video-exposure monitoring.

J Occup Health 2020 Jan;62(1):e12139

Department of Environmental Measurement and Control, University of Occupational and Environmental Health, Kitakyushu, Japan.

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.
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http://dx.doi.org/10.1002/1348-9585.12139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382765PMC
January 2020

Meeting Calorie and Protein Needs in the Critical Care Unit: A Prospective Observational Pilot Study.

Nutr Metab Insights 2020 26;13:1178638820905992. Epub 2020 Feb 26.

Clinical Sciences, Nestlé Health Science, Bridgewater Township, NJ. USA.

Background: Inadequate calorie and protein intake during critical illness is associated with poor clinical outcomes. Unfortunately, most critically ill patients do not consume adequate levels of these nutrients. An enteral formula with appropriate macronutrient composition may assist patients in meeting nutritional goals.

Design: This study was a single center, prospective, observational study of 29 adults in the medical intensive care unit who required enteral nutrition for at least 3 days. Subjects received a calorically dense, enzymatically hydrolyzed 100% whey peptide-based enteral formula for up to 5 days to assess the ability to achieve 50% of caloric goals within the first 3 days (primary outcome), the daily percentage of protein goals attained and gastrointestinal tolerance (secondary outcomes).

Result: A total of 29 subjects consented and began the study. Four subjects dropped out before first day and 25 subjects were included in analyses. Subjects were aged 55.5 ± 16.9 years with mean body mass index (BMI) of 27.9 ± 7.5 kg/m. Most (92%) subjects were on a mechanical ventilator and experienced organ failure. At least 50% of caloric and protein goals were achieved in 78.9% and 73.7% of the subjects, respectively, during the first 3 days. Overall, 75.0 ± 26.3% and 69.3 ± 26.7% of calorie and protein goals were achieved using the study formula.

Conclusions: Subjects fed enterally with a calorically dense, enzymatically hydrolyzed 100% whey peptide-based enteral formula exceeded 50% of caloric and protein goals in most critically ill subjects included in this study. Use of study formula did not lead to severe gastrointestinal intolerance.
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http://dx.doi.org/10.1177/1178638820905992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045298PMC
February 2020

Development of a measurement method to determine the ceiling exposure concentration of ortho-phthalaldehyde handling workers.

J Occup Health 2020 Jan 28;62(1):e12105. Epub 2019 Dec 28.

Department of Environmental Measurement and Control, University of Occupational and Environmental Health, Kitakyushu, Japan.

Objectives: The purpose of this research was to develop and validate an analytical method for rapid determination of the exposure of workers to ortho-phthalaldehyde (OPA) at the ceiling threshold concentration.

Methods: A 2,4-dinitrophenylhydrazine (DNPH)-silica cartridge was chosen as a sampler. OPA collected by the DNPH-silica cartridge was subsequently extracted with 5 mL of acetonitrile. A 50-µL aliquot of phosphoric acid/acetonitrile solution (2%, v/v) was added to 950 µL of the extraction solution and allowed to stand for 30 minutes at room temperature. This solution was then analyzed by high-performance liquid chromatography tandem mass spectrometry. The basic characteristics of the proposed method, such as recovery, repeatability, limit of quantification, and storage stability of the samples, were examined.

Results: The overall recoveries of OPA from OPA-spiked DNPH-silica cartridges were 93.6%-100.1% with relative standard deviations, representing the repeatability, of 1.5%-10.8%. The limit of quantification was 0.165 ng/sample. The recovery of OPA from DNPH-silica cartridges after 5 days of storage in a refrigerator exceeded 95%.

Conclusions: The proposed method enabled the determination of the OPA concentration corresponding to the Threshold Limit Value-Ceiling of 0.1 ppb recommended by the American Conference of Governmental Industrial Hygienists, with a minimum sampling time of 18 seconds (corresponding to a sampling volume of 300 mL at 25°C and 1 atm). Thus, this method will be useful for estimating worker exposures to OPA.
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http://dx.doi.org/10.1002/1348-9585.12105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970395PMC
January 2020

Direct methyl esterification with 2,2-dimethoxypropane for the simultaneous determination of urinary metabolites of toluene, xylene, styrene, and ethylbenzene by gas chromatography-mass spectrometry.

J Occup Health 2019 Jan;61(1):82-90

Osaka Occupational Health Service Center, Japan Industrial Safety and Health Association, Japan.

Objectives: The purpose of this study was to develop a simple and accurate gas chromatography-mass spectrometry (GC-MS) method for simultaneous determination of four urinary metabolites from four organic solvents, that is, hippuric acid (HA) from toluene, methylhippuric acid (MHA) from xylene, and mandelic acid (MA) and phenylglyoxylic acid (PGA) from styrene or ethylbenzene for biological monitoring.

Methods: The four metabolites were directly methyl-esterified with 2,2-dimethoxypropane and analyzed using GC-MS. The proposed method was validated according to the US Food and Drug Administration guidance. The accuracy of the proposed method was confirmed by analyzing a ClinChek -Control for occupational medicine (RECIPE Chemicals +Instruments GmbH).

Results: Calibration curves showed linearity in the concentration range of 10-1000 mg/L for each metabolite, with correlation coefficients >0.999. For each metabolite, the limits of detection and quantification were 3 mg/L and 10 mg/L, respectively. The recovery was 93%-117%, intraday accuracy, expressed as the deviation from the nominal value, was 92.7%-103.0%, and intraday precision, expressed as the relative standard deviation (RSD), was 1.3%-4.7%. Interday accuracy and precision were 93.4%-104.0% and 1.2%-9.5%, respectively. The analytical values of ClinChek obtained using the proposed method were sufficiently accurate.

Conclusions: The proposed method is a simple and accurate which is suitable for routine analyses that could be used for biological monitoring of occupational exposure to four organic solvents.
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http://dx.doi.org/10.1002/1348-9585.12026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499357PMC
January 2019

Very high-protein and low-carbohydrate enteral nutrition formula and plasma glucose control in adults with type 2 diabetes mellitus: a randomized crossover trial.

Nutr Diabetes 2018 08 30;8(1):45. Epub 2018 Aug 30.

Clinical Sciences, Nestlé Health Science, 1007 US Highway 202/206, Building JR2, Bridgewater, NJ, 08807, USA.

Background And Objectives: Standard enteral nutrition (EN) formulas can  worsen hyperglycemia in diabetic patients. We hypothesized that altering the proportion of macronutrients in a formula; increasing protein while decreasing carbohydrate concentrations would improve glycemic response. The objective of this study was to demonstrate that an EN formula containing a very high concentration of protein (in the form of whey peptides) and low concentration of carbohydrate provide better control of postprandial blood glucose relative to a very high-protein/higher-carbohydrate formula.

Subjects And Methods: This was a randomized crossover clinical trial of 12 ambulatory adult subjects with type 2 diabetes. The primary outcome was glycemic response following a bolus of isocaloric amounts of two EN formulas; the secondary outcome was insulin response. Subjects were randomized to the experimental or the control formula, on two separate days, 5-7 days apart.

Results: Mean blood glucose concentrations at 10-180 min post-infusion and mean area under the curve for glucose over 240 min post-infusion were significantly lower with the experimental formula than with the control formula (71.99 ± 595.18 and 452.62 ± 351.38, respectively; p = 0.025). There were no significant differences in the mean insulin concentrations over time, insulinogenic indices, and first-phase insulin measurements.

Conclusions: An EN formula containing high-protein and low-carbohydrate loads can significantly improve glucose control in subjects with type 2 diabetes in ambulatory settings as evidenced by observed improved glucose control without significant difference in insulin response.
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http://dx.doi.org/10.1038/s41387-018-0053-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115411PMC
August 2018

Concentration determination of urinary metabolites of N,N-dimethylacetamide by high-performance liquid chromatography-tandem mass spectrometry.

J Occup Health 2018 Mar 6;60(2):140-147. Epub 2017 Dec 6.

Department of Social Medicine, Saga University.

Objectives: N,N-Dimethylacetamide (DMAC) is widely used in industry as a solvent. It can be absorbed through human skin. Therefore, it is necessary to determine exposure to DMAC via biological monitoring. However, the precision of traditional gas chromatography (GC) is low due to the thermal decomposition of metabolites in the high-temperature GC injection port. To overcome this problem, we have developed a new method for the simultaneous separation and quantification of urinary DMAC metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Methods: Urine samples were diluted 10-fold in formic acid, and 1-μl aliquots were injected into the LC-MS/MS equipment. A C18 reverse-phase Octa Decyl Silyl (ODS) column was used as the analytical column, and the mobile phase consisted of a mixture of methanol and aqueous formic acid solution.

Results: Urinary concentrations of DMAC and its known metabolites (N-hydroxymethyl-N-methylacetamide (DMAC-OH), N-methylacetamide (NMAC), and S- (acetamidomethyl) mercapturic acid (AMMA) ) were determined in a single run. The dynamic ranges of the calibration curves were 0.05-5 mg/l (r≥0.999) for all four compounds. The limits of detection for DMAC, DMAC-OH, NMAC, and AMMA in urine were 0.04, 0.02, 0.05, and 0.02 mg/l, respectively. Within-run accuracies were 96.5%-109.6% with relative standard deviations of precision being 3.43%-10.31%.

Conclusions: The results demonstrated that the proposed method could successfully quantify low concentrations of DMAC and its metabolites with high precision. Hence, this method is useful for evaluating DMAC exposure. In addition, this method can be used to examine metabolite behaviors in human bodies after exposure and to select appropriate biomarkers.
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http://dx.doi.org/10.1539/joh.17-0098-OADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886881PMC
March 2018

Neuroinvasive West Nile Infection Elicits Elevated and Atypically Polarized T Cell Responses That Promote a Pathogenic Outcome.

PLoS Pathog 2016 Jan 21;12(1):e1005375. Epub 2016 Jan 21.

Benaroya Research Institute at Virginia Mason, Seattle, Washington, United States of America.

Most West Nile virus (WNV) infections are asymptomatic, but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death. Evidence from animal models suggests that neuroinvasive infections may arise as a consequence of impaired immune protection. However, other data suggest that neurologic symptoms may arise as a consequence of immune mediated damage. We demonstrate that elevated immune responses are present in neuroinvasive disease by directly characterizing WNV-specific T cells in subjects with laboratory documented infections using human histocompatibility leukocyte antigen (HLA) class II tetramers. Subjects with neuroinvasive infections had higher overall numbers of WNV-specific T cells than those with asymptomatic infections. Independent of this, we also observed age related increases in WNV-specific T cell responses. Further analysis revealed that WNV-specific T cell responses included a population of atypically polarized CXCR3+CCR4+CCR6- T cells, whose presence was highly correlated with neuroinvasive disease. Moreover, a higher proportion of WNV-specific T cells in these subjects co-produced interferon-γ and interleukin 4 than those from asymptomatic subjects. More globally, subjects with neuroinvasive infections had reduced numbers of CD4+FoxP3+ Tregs that were CTLA4 positive and exhibited a distinct upregulated transcript profile that was absent in subjects with asymptomatic infections. Thus, subjects with neuroinvasive WNV infections exhibited elevated, dysregulated, and atypically polarized responses, suggesting that immune mediated damage may indeed contribute to pathogenic outcomes.
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http://dx.doi.org/10.1371/journal.ppat.1005375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4721872PMC
January 2016

[Factors Affecting Outcomes of Repeated Penetrating Keratoplasty].

Nippon Ganka Gakkai Zasshi 2015 Sep;119(9):625-31

Purpose: To investigate prognosis for repeated penetrating keratoplasty (PKP) and factors that affect the outcome.

Methods: We retrospectively investigated graft survival rates, 1-year postoperative best-corrected visual acuity and irreversible rejection rates in 108 eyes of 106 patients that had repeated PKP. Factors that might affect the outcome were, age, number of previous PKP, original diseases, history of glaucoma and rejection and the use of postoperative immunosuppressant were also studied.

Results: Individual-factor analysis showed that history of rejection and postoperative immunosuppressant significantly increased the risk of postoperative rejection. Multi-factor analysis showed that graft survival rate was significantly lower among cases that had systemic immunosuppressants (steroids and cyclosporine). One year postoperative best-corrected visual acuity was significantly worse in cases that had history of glaucoma. In cases with history of rejection, systemic administration of postoperative immunosuppressants was significantly associated with postoperative irreversible rejection.

Conclusion: History of rejection and glaucoma tend to have poor outcome, and the outcome might not improve by postoperative immunosuppressants.
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September 2015

Applicability of concentrations obtained by working environment measurement to assessment of personal exposure concentrations of chemicals.

J Occup Health 2014 16;56(2):85-92. Epub 2014 Jan 16.

Kyushu Regional Safety and Health Service Center, Japan Industrial Safety and Health Association.

Objective: This study determined the applicability of Japanese working environment measurements to assessment of personal exposure concentrations of chemicals by comparing both levels of concentrations.

Methods: The chemicals measured in this study comprised eight kinds of vaporous chemicals as well as two kinds of chemicals in dust. Personal exposure measurements, Japanese working environment measurements and spot sampling measurements were undertaken in 70 companies.

Results: Personal exposure concentrations and the arithmetic mean value (EA2) of the working environment measurement concentrations obtained according to the Japanese working environment control system had statistically positive correlations (r=0.732-0.893, p<0.01) after logarithmic transformation. The 5th to 95th percentile values of personal exposure concentrations divided by EA2 ranged from 0.17 to 7.69 for vaporous chemicals and from 0.27 to 18.06 for dust. There was a relatively large difference between the personal exposure concentrations and the EA2 obtained in weighing, forming and bonding use-processes. In such cases, the B-value measured in ten minutes in the Japanese working environment control system, which is almost the same as the spot measurement concentration in this study, is supposed to be substituted for the EA2 value.

Conclusions: Ten times the EA2 of the working environment measurement concentrations, or ten times the B-value, obtained according to the Japanese working environment control system can be used to conservatively estimate the personal exposure concentrations in EU workplaces as well as in occupational exposure scenarios of the Regulation on Registration, Evaluation, Authorisation and Restriction of Chemicals.
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http://dx.doi.org/10.1539/joh.12-0243-oaDOI Listing
June 2015

Simultaneous extraction of acidic and basic drugs from urine using mixed-mode monolithic silica spin column bonded with octadecyl and cation-exchange group.

J Sep Sci 2011 Aug 6;34(16-17):2232-9. Epub 2011 Jun 6.

Department of Forensic Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.

A method coupling spin column extraction with gas chromatography-mass spectrometry was developed for the simultaneous extraction of acidic and basic drugs from urine. Benzodiazepines, local anaesthetics, antidepressants, and barbiturates were used as model drugs. Sample loading, washing, and elution of the target drugs were accomplished by centrifugation of the column. In this study, mixed-mode monolithic silica bonded with a C18 reversed-phase and a strong cation exchange phase was packed in a spin column. The pH of a urine sample (0.2 mL) was adjusted to 3 and the analytes adsorbed onto the column were eluted with 0.1 mL of MeOH containing 2% NH3; all the tested drugs were simultaneously extracted from urine. The recovery of the tested drugs was 65-123%. Up to a concentration of 2500 ng/mL of the target drugs in urine, a linear curve was observed (r(2)>0.996). The intra- and interday RSDs at three different concentrations in urine were 2.1-14.7%. For RSDs lower than 15%, the limits of detection were 1-25 ng/mL. The proposed method was successfully applied for clinical and forensic cases and the results thus obtained were in good agreement with those obtained by conventional methods.
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http://dx.doi.org/10.1002/jssc.201100165DOI Listing
August 2011

Determination method for xylidines in workplace air.

J Occup Health 2011 16;53(3):230-3. Epub 2011 Mar 16.

Osaka Occupational Health Service Center, Japan Industrial Safety and Health Association, Japan.

Objectives: The purpose of this research was to develop a determination method for xylidines (XLDs) in workplace air for risk assessment.

Methods: The characteristics of the proposed method, such as recovery, detection limit, reproducibility, and storage stability of the samples were examined.

Results: An air sampler cassette containing two sulfuric acid-treated glass fiber filters was chosen as the sampler. The XLDs were extracted from the sampler filters, derivatized with heptafluorobutyric anhydride, and then analyzed by a gas chromatograph equipped with a mass spectrometer. The average recoveries of XLDs from the spiked sampler were 83-101% for personal exposure monitoring. The recovery after 5 days of storage in a refrigerator exceeded 90%. The overall limit of quantitation (LOQ) was 0.600 g/sample. The relative standard deviation, which represents the overall reproducibility defined as precision, was 0.8-10.3%.

Conclusions: The proposed method enables 4-hour personal exposure monitoring of XLDs at concentrations equaling 0.001-2 times the threshold limit value-time-weighted average (TLV-TWA: 0.5 ppm) adopted by the American Conference of Governmental Industrial Hygienists, and is useful for estimating worker exposure to XLDs.
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http://dx.doi.org/10.1539/joh.10-0003-brDOI Listing
October 2011

Direct determination of N-methyl-2-pyrrolidone metabolites in urine by HPLC-electrospray ionization-MS/MS using deuterium-labeled compounds as internal standard.

J Chromatogr B Analyt Technol Biomed Life Sci 2009 Nov 28;877(29):3743-7. Epub 2009 Aug 28.

Research Center for Occupational Poisoning, Tokyo Rosai Hospital, Japan Labour Health and Welfare Organization, 4-13-21, Ohmori-Minami, Ohta-ku, Tokyo 143-0013, Japan.

N-methyl-2-pyrrolidone (NMP) has been used in many industries and biological monitoring of NMP exposure is preferred to atmospheric monitoring in occupational health. We developed an analytical method that did not include solid phase extraction (SPE) but utilized deuterium-labeled compounds as internal standard for high-performance liquid chromatography-electrospray ionization-mass spectrometry using a C30 column. Urinary concentrations of NMP and its known metabolites 5-hydoxy-N-methyl-2-pyrrolidone (5-HNMP), N-methyl-succinimide (MSI), and 2-hydroxy-N-methylsuccinimide (2-HMSI) were determined in a single run. The method provided baseline separation of these compounds. Their limits of detection in 10-fold diluted urine were 0.0001, 0.006, 0.008, and 0.03 mg/L, respectively. Linear calibration covered a biological exposure index (BEI) for urinary concentration. The within-run and total precisions (CV, %) were 5.6% and 9.2% for NMP, 3.4% and 4.2% for 5-HNMP, 3.7% and 6.0% for MSI, and 6.5% and 6.9% for 2-HMSI. The method was evaluated using international external quality assessment samples, and urine samples from workers exposed to NMP in an occupational area.
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http://dx.doi.org/10.1016/j.jchromb.2009.08.036DOI Listing
November 2009

Determination of dimethyl sulfoxide and dimethyl sulfone in urine by gas chromatography-mass spectrometry after preparation using 2,2-dimethoxypropane.

Biomed Chromatogr 2010 May;24(5):465-71

Osaka Occupational Health Service Center, Japan Industrial Safety and Health Association, Tosabori, Nishi-ku, Osaka, Japan.

A method for routinely determination of dimethyl sulfoxide (DMSO) and dimethyl sulfone (DMSO(2)) in human urine was developed using gas chromatography-mass spectrometry. The urine sample was treated with 2,2-dimethoxypropane (DMP) and hydrochloric acid for efficient removal of water, which causes degradation of the vacuum level in mass spectrometer and shortens the life-time of the column. Experimental DMP reaction parameters, such as hydrochloric acid concentration, DMP-urine ratio, reaction temperature and reaction time, were optimized for urine. Hexadeuterated DMSO was used as an internal standard. The recoveries of DMSO and DMSO(2) from urine were 97-104 and 98-116%, respectively. The calibration curves showed linearity in the range of 0.15-54.45 mg/L for DMSO and 0.19-50.10 mg/L for DMSO(2). The limits of detection of DMSO and DMSO(2) were 0.04 and 0.06 mg/L, respectively. The relative standard deviations of intra-day and inter-day were 0.2-3.4% for DMSO and 0.4-2.4% for DMSO(2). The proposed method may be useful for the biological monitoring of workers exposed to DMSO in their occupational environment.
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http://dx.doi.org/10.1002/bmc.1313DOI Listing
May 2010

Optimization of the determination of ortho-phthalaldehyde in air by derivatization with 2,4-dinitorophenylhyrazine (DNPH).

J Occup Health 2009 18;51(4):386-90. Epub 2009 Jun 18.

Chugoku and Shikoku Regional Safety and Health Service Center, Japan Industrial Safety and Health Association, Japan.

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http://dx.doi.org/10.1539/joh.m9005DOI Listing
December 2009

Analysis of the varicella-zoster virus IE62 N-terminal acidic transactivating domain and its interaction with the human mediator complex.

J Virol 2009 Jun 8;83(12):6300-5. Epub 2009 Apr 8.

Departments of Microbiology and Immunology, University at Buffalo SUNY, Buffalo, New York 14214, USA.

The varicella-zoster virus major transactivator, IE62, contains a potent N-terminal acidic transcriptional activation domain (TAD). Our experiments revealed that the minimal IE62 TAD encompasses amino acids (aa) 19 to 67. We showed that the minimal TAD interacts with the human Mediator complex. Site-specific mutations revealed residues throughout the minimal TAD that are important for both activation and Mediator interaction. The TAD interacts directly with aa 402 to 590 of the MED25 subunit, and site-specific TAD mutations abolished this interaction. Two-dimensional nuclear magnetic resonance spectroscopy revealed that the TAD is intrinsically unstructured. Our studies suggest that transactivation may involve the TAD adopting a defined structure upon binding MED25.
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http://dx.doi.org/10.1128/JVI.00054-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2687382PMC
June 2009