N-Terminal Amino Acid Affects the Translation Efficiency at Lower Temperatures in a Reconstituted Protein Synthesis System.

The Escherichia coli (E. coli)-based protein synthesis using recombinant elements (PURE) system is a cell-free protein synthesis system reconstituted from purified factors essential for E. coli translation. The PURE system is widely used for basic and synthetic biology applications. One of the major challenges associated with the PURE system is that the protein yield of the system varies depending on the protein. Studies have reported that the efficiency of translation is significantly affected by nucleotide and amino acid sequences, especially in the N-terminal region. Here, we investigated the inherent effect of various N-terminal sequences on protein synthesis using the PURE system. We found that a single amino acid substitution in the N-terminal region significantly altered translation efficiency in the PURE system, especially at low temperatures. This result gives us useful suggestions for the expression of the protein of interest in vitro and in vivo.

Occupation- and industry-specific cancer mortality among Japanese women from 1980 to 2015.

BACKGROUND: The number of women in Japan who continue working after childbirth is on the rise. Over the past few years, Japan's cancer mortality rate has increased. About 50% of all cancer deaths among Japanese women aged 25-64 are caused by lung, gastric, pancreatic and colorectal cancers. This study aims to examine the difference in mortality risk for key cancers among women and explore the effect of the economic crisis in the mid-1990s separately for occupational and industrial categories. METHODS: Data from 1980 to 2015 were gathered from the Japanese Population Census and National Vital Statistics conducted in the same year. A Poisson regression analysis was used to estimate mortality risk and mortality trends for lung, gastric, pancreatic and colorectal cancer among Japanese working women aged 25-64 years. RESULTS: Across most industrial and occupational groups, the trends in age-standardised cancer mortality rate for women have declined. Workers in management, security and transportation have a higher cancer mortality risk than sales workers. The risk of death from all four cancers is higher for workers in the mining and electricity industries than for wholesale and retail workers. CONCLUSION: To improve the health and well-being of employed Japanese women, it is crucial to monitor cancer mortality trends. Using these population-level quantitative risk estimates, industry- and occupation-specific prevention programmes can be developed to target women at higher cancer risk and enable the early detection and treatment of cancer.

Difference in Mortality Rates by Occupation in Japanese Male Workers Aged 25 to 64 Years from 1980 to 2015.

This study examines the trends in mortality among Japanese working men, across various occupational categories, from 1980 to 2015. A Poisson model of trend, occupational category, and step variable was analysed for eight occupational categories separately, by cause, to explore the trends in mortality. This study found a sharp increase in mortality in the late 1990s, especially among professionals and managers. The overall trends in cancer, ischemic heart disease (IHD), cerebrovascular disease (CVD), and suicide mortality decreased across almost all occupational categories from 1980 to 2015, although there was an increasing trend in cancer of 0.5% among managers. Clerical workers had the greatest relative decrease in mortality rates from cancer (-82.9%), IHD (-81.7%), and CVD (-89.1%). Japan continues to make gains in lowering mortality and extending life expectancy, but its workplace culture must improve to ensure that those working at the heart of the Japanese corporate world can also benefit from Japan's progress in health. Mortality rates in working-aged Japanese men have been declining. However, similar declines are not evident among managers, for whom the mortality rate is remaining stable or slightly increasing. There is a need to address the needs of managers and improve workplace environments for these workers.

Mortality risks among blue- and white-collar workers: A time series study among Japanese men aged 25-64 years from 1980 to 2015.

OBJECTIVE: We aimed to analyse age-standardised mortality trends in Japan among blue- and white-collar male workers aged 25-64 years, by major causes of mortality from 1980 to 2015. METHODS: Five-yearly mortality data were extracted from occupation-specific vital statistics maintained by the Japanese Ministry of Health, Labour and Welfare. A time series study was conducted among employed men aged 25-64 years. Age-standardised mortality trends by occupational category were calculated separately for all cancers, ischaemic heart disease, cerebrovascular disease and suicide. Poisson regression analysis was performed to analyse mortality trends by occupational category for each cause. RESULTS: Mortality rates for all cancers and ischaemic heart disease were higher among white-collar workers than blue-collar workers throughout the 35-year study period. The gap in the mortality rates for all four causes of death among blue- and white-collar workers widened in 2000 after Japan's economic bubble burst in the late 1990s. Simultaneously, suicide mortality rates among white-collar workers increased sharply and have remained higher than among blue-collar workers. CONCLUSIONS: White-collar male workers in Japan have a higher risk of mortality than male blue-collar workers. However, despite substantial differences, significant progress has been made in recent years in reducing mortality across all occupations in Japan.

HBV- and HCV- infected workers in the Japanese workplace.

Around three million Japanese are persistently infected with HBV or HCV. Though most of them work in various industries, little is known about the actual conditions in their workplaces. To clarify the workplace conditions of workers with hepatitis, three kinds of questionnaire surveys, answered by occupational health physicians and workers with hepatitis, were carried out. The rates of workers recognized as workers with hepatitis B or C by occupational health physicians were 0.82% and 0.48% of 130,092 workers, respectively. About 30% of workers with hepatitis were engaged in "hazardous work". The percentage of workers engaged in various types of hazardous work among workers with hepatitis was nearly the same as that among all Japanese workers. About 30% of occupational health physicians witnessed exacerbation of hepatitis in the workers at their workplaces, and 22% of workers with hepatitis experienced exacerbation of hepatitis. The rate of workers with hepatitis who had experienced exacerbation was not significantly different between workers with and without hazardous work. Workers with hepatitis have strong concerns about the relationship between work and exacerbation. As causes of exacerbation, occupational health physicians cited "unknown", "drinking" and "quit treatment" while workers with hepatitis answered "work-related causes", besides "unknown" and "drinking."

[International comparison of sensitizing chemical substances].

Some occupational and environmental chemicals cause allergic diseases. To prevent chemical allergies, it is essential to identify the chemical substances that cause sensitization and to eliminate such sensitizers from daily life. As an occupational countermeasure, information for evaluating sensitization of chemical substances is needed. The aims of this article are to compare the criteria for sensitizers among national organizations in various countries and international organizations, and to make out a list of these chemical substances. The definition of sensitizing chemicals and the designation of respective sensitizers according to the PRTR law, Japan Society for Occupational Health (JSHO), American Conference of Governmental Industrial Hygienists (ACGIH), European Union (EU), Deutsche Forschungsgemeinshaft (DFG) and Japanese Society of Occupational and Environmental Allergy were studied. There are 1,389 chemical substances which are designated as sensitizers by any of the laws and five organizations. We specify each chemical substance in the list.

Hemoglobin adducts as a marker of exposure to chemical substances, especially PRTR class I designated chemical substances.

Many new biomarkers are being studied, in addition to classical biomarkers, such as chemical substances and their metabolites in blood and urine and modified enzymes. Among these new biomarkers, hemoglobin adducts are thought to be especially useful for the estimation of chemical exposures. We review here the use of biomarkers for monitoring exposures to nine substances, mainly focusing on PRTR class I designated chemical substances, styrene, phenyloxirane (styrene oxide), 4,4'-methylendiphenyl diisocyanate (MDI), 4,4'-methylendianiline (MDA), 1,3-butadiene, ethylene oxide, propylene oxide, acrylamide and acrylonitrile. Hemoglobin adduct levels were elevated after exposures to styrene, MDI, MDA, 1, 3-butadiene, ethylene oxide, acrylamide and acrylonitrile. Moreover, hemoglobin adducts of butadiene, ethylene oxide, acrylamide and acrylonitrile have several useful advantages. For example, the hemoglobin adduct of 1,3-butadiene is an even more useful biomarker of exposure than urinary metabolites, and in the case of ethylene oxide, even though the concentration of ethylene oxide-Hb in the blood of workers did not exceed the value of the German exposure equivalent, a significant difference in it was found between workers and a control group. Also hemoglobin adducts of acrylamide and acrylonitrile can reflect their exposures because there are no urinary metabolites of acrylamide and acrylonitrile that are useful for exposure assessment. In addition to these advantages, hemoglobin adducts are superior to DNA adducts with respect to the availability of large amounts, availability of methods for chemical identification, and well-defined life spans due to the absence of repair. Hemoglobin adducts can be effective biomarkers for assessing exposure to and the effects of chemicals.

Paired acute inhalation test reveals that acetaldehyde toxicity is higher in aldehyde dehydrogenase 2 knockout mice than in wild-type mice.

Aldehyde dehydrogenase 2 (ALDH2) is an important enzyme that oxidizes acetaldehyde. Approximately 45% of Chinese and Japanese individuals have the inactive ALDH2 genotypes (ALDH2*2/*2 and ALDH2*1/*2); acute inhalation toxicity of acetaldehyde has not been evaluated in these populations. We compared the toxicity between wild-type (Aldh2+/+) and Aldh2-inactive transgenic (Aldh2-/-) mice by using the paired acute inhalation test modified from the acute toxic class method (OECD TG433). Blood acetaldehyde level was measured 4 hr after the inhalation. A pair of Aldh2+/+ and Aldh2-/- mice was put into a chamber and was exposed to 5000 ppm of acetaldehyde. At the start of the inhalation, the mice exhibited hypoactivity and closing of the eyes. Subsequently, symptoms such as crouching, bradypnea, and piloerection were observed. Flushing was observed only in the Aldh2+/+ mice. Symptoms such as tears, straggling gait, prone position, pale skin, abnormal deep respiration, dyspnea, and one case of death were observed only in the Aldh2-/- mice. The symptoms did not change 1 hr after inhalation in the Aldh2+/+ mice. In contrast, in the Aldh2-/- mice, the symptoms became more severe until the end of the inhalation. The blood acetaldehyde level in the Aldh2-/- mice was approximately twice that in the Aldh2+/+ mice 4 hr after inhalation. The Aldh2-/- mice evidently showed more severe toxicity as compared with the Aldh2+/+ mice due to acute inhalation of acetaldehyde at a concentration of 5000 ppm. Acetaldehyde toxicity in Aldh2+/+ and Aldh2-/- mice was estimated and classified one class different. Based on this study, acetaldehyde inhalations were inferred to pose a higher risk to ALDH2-inactive human individuals.

Human GM-CSF induces HIV-1 LTR by multiple signalling pathways.

Human immunodeficiency virus type-1 (HIV-1) gene expression is known to be affected by numerous cytokines or growth factors. However, the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on long terminal repeat (LTR)-mediated transcription of HIV-1 still remains unknown. By transient transfection experiments with HIV-1 LTR reporter constructs, we showed that strong LTR-mediated activation was induced by GM-CSF in mouse Ba/F3 cells expressing human GM-CSF receptors (GM-CSFR). Mutational analysis of the HIV-1 LTR reporters revealed that both NF-kappaB and Sp1 binding sites play important roles as positive regulatory elements. Analysis of various mutants of the cytoplasmic region of GM-CSFR indicated that both the conserved membrane proximal region and tyrosine residues located in the distal part of the beta subunit were required for HIV-1 LTR activation. Possible involvement of MAPK and PI3-K signalling pathways was suggested by the partial inhibition by wortmannin, a specific inhibitor of the PI3-K pathway, and enhancement by constitutively active MEK1, of HIV-1 LTR activation. However, the MEK1 pathway is not essential since MEK1 inhibitor PD98059 did not suppress GM-CSF-induced HIV-1-LTR activation. Further analyses of GM-CSFR mutants suggested that some other unknown signalling pathway also participates in GM-CSF-induced HIV-1 LTR activation. Taken together, the data suggest that GM-CSF could upregulate the LTR-driven transcription of HIV-1 through modulation of NF-kappaB and SP1 by multiple signalling pathways.

International comparison of criteria for evaluating sensitization of PRTR-designated chemical substances.

In this study, we aim to compare the criteria for sensitizers among national organizations in various countries and international organizations, and to specify whether each Pollutant Release and Transfer Register (PRTR)-designated chemical substance is a sensitizer by each organization. The definition of sensitizing chemicals and the designation of respective sensitizers according to the PRTR law, Japan Society for Occupational Health (JSOH), American Conference of Governmental Industrial Hygienists (ACGIH), European Union (EU), and Deutsche Forschungsgemeinshaft (DFG) were studied. Of the 435 PRTR-designated chemical substances, 15 are listed as sensitizers according to the PRTR law, 16 as sensitizers of the airway and 21 as sensitizers of the skin by JSOH, 12 as sensitizers (no discrimination) by ACGIH, 19 (airway) and 85 (skin) by EU, and 15 (airway) and 43 (skin) by DFG. Only 9 substances were designated as sensitizers by all these organizations. The variation in the designation of sensitizers is accounted for by the differences in the classification criteria and grouping of chemical substances. JSOH limits the definition of sensitizers to substances that induce allergic reactions in humans and uses only human data. Other organizations utilize not only human evidence but also appropriate animal tests. In addition, EU designates an isocyanate as a sensitizer except those for which there is evidence showing that they do not cause respiratory sensitivity. The worldwide enforcement of the globally harmonized system (GHS) of classification and labeling of chemicals could promote not only the consistent designation of sensitizers among national and international organizations, but also the development of testing guidelines and classification criteria for mixtures.

Evidence-based prevention (EBP): A review of cytochrome P450 expression in the bronchial epithelium and new approach to lung cancer prevention.

The number of fatalities in Japan attributable to lung cancer exceeded 50000 in 2001. It is socially desirable that various markers, which can be utilized for the prevention of lung cancer, be established. We believe that smoking or exposure to carcinogens in air induces mutations in bronchial and alveolar epithelia, leading to the development of lung cancer. It would be useful to have markers of individual differences in susceptibility to chemical carcinogen-induced lung cancer 1) to identify genetic polymorphisms of enzymes metabolizing chemical carcinogens and 2) to investigate the expression of enzymes metabolizing chemical carcinogens. In this paper, we review CYP expression in the bronchial epithelium. CYP1, CYP2 and CYP3 are expressed in the bronchial epithelium. We also show the relationship between the genetic polymorphisms of cytochrome P450 (CYP) and a person's susceptibility to chemical carcinogen-induced lung cancer. We demonstrate the relationship between cigarette consumption and the CYP expression profile in the bronchial epithelium. To maintain and promote public health, we must apply evidence, such as CYP polymorphisms and CYP profiles to disease prevention and also to aggressively advance evidence-based prevention (EBP) of lung cancer.

Real-time monitoring of conformational dynamics of the epsilon subunit in F1-ATPase.

It has been proposed that C-terminal two alpha-helices of the epsilon subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F(1) from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the gamma subunit and C terminus of the epsilon subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5'-Adenyl-beta,gamma-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic beta subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the epsilon subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 degrees C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated epsilon subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 degrees C reached to 0.67 mm, a consistent value to assume that the epsilon subunit acts as a sensor of ATP concentration in the cell.

F0F1-ATPase/synthase is geared to the synthesis mode by conformational rearrangement of epsilon subunit in response to proton motive force and ADP/ATP balance.

The epsilon subunit in F0F1-ATPase/synthase undergoes drastic conformational rearrangement, which involves the transition of two C-terminal helices between a hairpin "down"-state and an extended "up"-state, and the enzyme with the up-fixed epsilon cannot catalyze ATP hydrolysis but can catalyze ATP synthesis (Tsunoda, S. P., Rodgers, A. J. W., Aggeler, R., Wilce, M. C. J., Yoshida, M., and Capaldi, R. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6560-6564). Here, using cross-linking between introduced cysteine residues as a probe, we have investigated the causes of the transition. Our findings are as follows. (i) In the up-state, the two helices of epsilon are fully extended to insert the C terminus into a deeper position in the central cavity of F1 than was thought previously. (ii) Without a nucleotide, epsilon is in the up-state. ATP induces the transition to the down-state, and ADP counteracts the action of ATP. (iii) Conversely, the enzyme with the down-state epsilon can bind an ATP analogue, 2',3'-O-(2,4,6-trinitrophenyl)-ATP, much faster than the enzyme with the up-state epsilon. (iv) Proton motive force stabilizes the up-state. Thus, responding to the increase of proton motive force and ADP, F0F1-ATPase/synthase would transform the epsilon subunit into the up-state conformation and change gear to the mode for ATP synthesis.