Overexpression of Orange Gene (OsOr-R115H) Enhances Heat Tolerance and Defense-Related Gene Expression in Rice (Oryza sativa L.).

In plants, the orange (Or) gene plays roles in regulating carotenoid biosynthesis and responses to environmental stress. The present study investigated whether the expression of rice Or (OsOr) gene could enhance rice tolerance to heat stress conditions. The OsOr gene was cloned and constructed with OsOr or OsOr-R115H (leading to Arg to His substitution at position 115 on the OsOr protein), and transformed into rice plants. The chlorophyll contents and proline contents of transgenic lines were significantly higher than those of non-transgenic (NT) plants under heat stress conditions. However, we found that the levels of electrolyte leakage and malondialdehyde in transgenic lines were significantly reduced compared to NT plants under heat stress conditions. In addition, the levels of expression of four genes related to reactive oxygen species (ROS) scavenging enzymes (OsAPX2, OsCATA, OsCATB, OsSOD-Cu/Zn) and five genes (OsLEA3, OsDREB2A, OsDREB1A, OsP5CS, SNAC1) responded to abiotic stress was showed significantly higher in the transgenic lines than NT plants under heat stress conditions. Therefore, OsOr-R115H could be exploited as a promising strategy for developing new rice cultivars with improved heat stress tolerance.

Multiple cytochrome P450 isoforms are involved in the generation of a pharmacologically active thiol metabolite, whereas paraoxonase 1 and carboxylesterase 1 catalyze the formation of a thiol metabolite isomer from ticlopidine.

Ticlopidine is a first-generation thienopyridine antiplatelet drug that prevents adenosine 5'-diphosphate (ADP)-induced platelet aggregation. We identified the enzymes responsible for the two-step metabolic bioactivation of ticlopidine in human liver microsomes and plasma. Formation of 2-oxo-ticlopidine, an intermediate metabolite, was NADPH dependent and cytochrome P450 (CYP) 1A2, 2B6, 2C19, and 2D6 were involved in this reaction. Conversion of 2-oxo-ticlopidine to thiol metabolites was observed in both microsomes (M1 and M2) and plasma (M1). These two metabolites were considered as isomers, and mass spectral analysis suggested that M2 was a thiol metabolite bearing an exocyclic double bond, whereas M1 was an isomer in which the double bond was migrated to an endocyclic position in the piperidine ring. The conversion of 2-oxo-ticlopidine to M1 in plasma was significantly increased by the addition of 1 mM CaCl2. In contrast, the activity in microsomes was not changed in the presence of CaCl2. M1 formation in plasma was inhibited by EDTA but not by other esterase inhibitors, whereas this activity in microsomes was substantially inhibited by carboxylesterase (CES) inhibitors such as bis-(p-nitrophenyl)phosphate (BNPP), diisopropylphosphorofluoride (DFP), and clopidogrel. The conversion of 2-oxo-ticlopidine to M1 was further confirmed with recombinant paraoxonase 1 (PON1) and CES1. However, M2 was detected only in NADPH-dependent microsomal incubation, and multiple CYP isoforms were involved in M2 formation with highest contribution of CYP2B6. In vitro platelet aggregation assay demonstrated that M2 was pharmacologically active. These results collectively indicated that the formation of M2 was mediated by CYP isoforms whereas M1, an isomer of M2, was generated either by human PON1 in plasma or by CES1 in the human liver.

Inter-individual variability in OCT1 expression and its relationship with OCT1 genotype in liver samples from a Korean population.

To clarify inter-individual variation in the expression of organic cation transporter 1 (OCT1), the levels of OCT1 mRNA and protein from 65 human liver samples were examined by real-time PCR and Western blot analysis and were associated with OCT1 genotypes. The expression levels of OCT1 mRNA and protein in 65 liver samples of Korean origin were not normally distributed and varied by 23.6- and 15.9-fold, respectively. OCT1 mRNA expression was correlated with OCT1 protein expression with a correlation coefficient of 0.641 (p < 0.0001). However, non-genetic factors, such as age, gender, and cholestasis, were not significantly associated with OCT1 expression. When quantitative expression levels were compared in relation to OCT1 promoter SNPs, there was no significant difference in OCT1 expression levels among the -1795 GG, GA, and AA genotypes. Moreover, expression levels of OCT1 were not changed in relation to the -1756 genotypes. Inter-individual variation in OCT1 mRNA and protein expression levels in the liver did not correlate with OCT1 genotypes or non-genetic factors, such as age, gender, and cholestasis. These results suggest that genetic and non-genetic factors may not be a significant contributing factor of variations in OCT1 expression from liver samples of Korean origin.

Identification of a null allele of cytochrome P450 3A7: CYP3A7 polymorphism in a Korean population.

Cytochrome P450 3A7 (CYP3A7) is expressed in the human fetal liver and plays a role in the metabolism of hormones, drugs, and toxic compounds. Genetic variants of CYP3A7 are associated with serum estrone level, bone density, and hepatic CYP3A activity in adults. We analyzed the genetic variations of CYP3A7 in a Korean population. From direct sequencing of all exons and flanking regions of the CYP3A7 gene in 48 Koreans, we found five genetic variants, including three novel variants. One variant, a thymidine insertion in exon 2 (4011insT), causes premature termination of CYP3A7 translation, which may result in a null phenotype. The novel variant was assigned to the CYP3A7*3 allele by the CYP allele nomenclature committee. For further screen of this novel variant in other ethnic populations, we used pyrosequencing to analyze an additional 185 Koreans, 100 African Americans, 100 Caucasians, and 159 Vietnamese for the presence of this variant. The variant was not found in any other individuals, except for one Korean subject. The frequencies of two known functional alleles, CYP3A7*2 and CYP3A7*1C, were 26 and 0%, respectively, in Koreans. The frequencies of the functional CYP3A7 polymorphisms in Koreans were significantly different from those in Caucasians and African Americans. This is the first report of a null-type allele of the CYP3A7 gene. It also provides population-level genetic data on CYP3A7 in Koreans to reveal the wide ethnic variation in CYP3A7 polymorphism.

Subjective ratings of upper extremity exposures: inter-method agreement with direct measurement of exposures.

This study examined the agreement of subjective ratings of upper extremity exposures with corresponding direct measurements obtained simultaneously from workers. Psychophysical ratings of exposure, based on the Borg CR-10 scale, were obtained for the period of time in which direct measurements were acquired using electrogoniometers (wrist), electroinclinometers (shoulder) and electromyography (grip force). Subjects were selected from workers at two automobile manufacturing plants. Significant relationships between subjective ratings of wrist position and measured wrist posture or motion and between ratings of shoulder position and measured shoulder posture were not found. Ratings of manual effort were significantly correlated with directly measured grip force (% maximum voluntary contraction). Ratings of pace were significantly correlated with directly measured wrist motion and this relationship was strengthened with the addition of relative grip force as a covariate. Workers with hand/wrist symptoms provided ratings that were more strongly related to the directly measured exposures than those without symptoms. Self-report by workers is an alternative to more resource-intensive and invasive exposure assessment methods. However, the validity of workers' self-reported exposure assessments has been questioned. The objective of this study was to examine the agreement of selected questionnaire items with corresponding direct measurements from bioinstrumentation and to provide a better understanding of worker self-reports.

Detection and kinetics of mucosal pathogenic bacteria binding with polysaccharides.

The detection and kinetics of mucosal pathogenic bacteria binding on polysaccharide ligands were studied using a surface plasmon resonance biosensor. The kinetic model applied curve-fitting to the experimental surface plasmon resonance sensorgrams to evaluate the binding interactions. The kinetic parameters for the mucosal pathogenic bacteria (Pseudomonas aeruginosa, Pseudomonasfluorescens, Serratia marcescens) with the alginate ligand were determined from a kinetic model. In addition, the binding interactions of the mucosal pathogenic bacteria with polysaccharide binding pairs (Pseudomonas aeruginosa/alginate, Streptococcus pneumoniae/pneumococcal polysaccharide, Staphylococcus aureus/pectin) were also compared with their kinetic parameters. The rate constants of association for Pseudomonas aeruginosa with the alginate ligand were higher than those for Pseudomonas fluorescens. Serratia marcescens had no detectable interaction with the alginate ligand. The adhesion affinity of Pseudomonas aeruginosa with alginate was higher than that for the other binding pairs. The binding affinities of the pathogenic bacteria with their own polysaccharide were higher than that of Staphylococcus aureus with pectin. Measuring the contact angle was found to be a feasible method for detecting binding interactions between analytes and ligands.

Medium- and long-term reproducibility of self-reported exposure to physical ergonomics factors at work.

INTRODUCTION: The literature is sparse on reproducibility of self-reported exposure to physical ergonomics risk factors for musculoskeletal disorders (MSDs). Aims of this study were to evaluate, in a cohort of workers interviewed up to three times: 1-year test-retest reliability; and 5- and 6-year recall of physical exposures. We also examined whether reproducibility was influenced by the presence of UE MSD or by technological changes introduced between the last two surveys. METHODS: A cohort of automobile manufacturing employees was interviewed at baseline, one and six years later about work history, physical and psychosocial exposures at work, upper limb symptoms, injury and medical history, and demographics. Agreement between interviews was evaluated by intraclass correlation and Spearman coefficients. Differences in exposure between 1- and 6-year follow-up were analyzed by Wilcoxon matched-pairs signed-ranks test. RESULTS: Large and significant decreases in work pace and physical effort were observed from baseline, although an upper extremity composite index was quite stable in the total population. One-year test-retest reliability was fair to good for the composite exposure index (ICC=0.58), whole-body vibration, handling parts, and tool use, but poor for the other variables considered. Long-term reproducibility, from baseline or 1-year follow-up to 6-year follow-up, was poor for the composite index and almost all single items. UE MSD case status influenced 1-year test-retest reliability, with subjects who changed case status from baseline displaying higher reliability, but not reproducibility of recalled exposures. A strong regression to the mean effect was observed on exposures reported at follow-up surveys. CONCLUSIONS: Recalled ergonomics exposures could be employed in retrospective cohort studies as a somewhat reliable and unbiased estimate of the self-reported exposures that would have been obtained up to one year earlier, but not over a longer period (5-6 years). These longer-term results may have been limited by difficulty in matching jobs between interviews; also the regression to the mean effect likely contributed to reduce agreement. Changes in production technology and work organization produced a decrease in physical workload intensity and job pace, but did not have a substantial impact on an exposure index for the upper limb.

A surface plasmon resonance biosensor for detecting Pseudomonas aeruginosa cells with self-assembled chitosan-alginate multilayers.

A self-assembled multilayer (SAMu) including the alginate layer was prepared for detecting Pseudomonas aeruginosa cells in a solution and its potential was evaluated with a BIAcore system. After layer-by-layer formation, the refractive units (RU) values monitored with the biosensor increased by the interaction between the layers. The responses by the binding of P. aeruginosa cells to the alginate-immobilized SAMu were visualized immediately upon injection of the cell suspension. The RU values after injection of the cells were measured with approximately 1152, 656 and 173 for 1x10(9), 1x10(8) and 1x10(7)CFU/ml. This result suggests that the alginate-immobilized SAMu will have useful application for detecting P. aeruginosa cells in a biosensor analysis.

Work routinization and implications for ergonomic exposure assessment.

Jobs in many modern settings, including manufacturing, service, agriculture and construction, are variable in their content and timing. This prompts the need for exposure assessment methods that do not assume regular work cycles. A scheme is presented for classifying levels of routinization to inform development of an appropriate exposure assessment strategy for a given occupational setting. Five levels of routinization have been defined based on the tasks of which the job is composed: 1) a single scheduled task with a regular work cycle; 2) multiple cyclical tasks; 3) a mix of cyclical and non-cyclical tasks; 4) one non-cyclical task; 5) multiple non-cyclical tasks. This classification, based primarily on job observation, is illustrated through data from a study of automobile manufacturing workers (n = 1200), from which self-assessed exposures to physical and psychosocial stressors were also obtained. In this cohort, decision latitude was greater with higher routinization level (p < 0.0001), and the least routinized jobs showed the lowest self-reported exposure to physical ergonomic stressors. The job analysis checklist developed for non-routinized jobs is presented, and limitations of the task analysis method utilized in the study are discussed. A work sampling approach to job analysis is recommended as the most efficient way to obtain a comparable unbiased exposure estimate across all routinization levels.

D-threo-tetrahydrobiopterin is synthesized via 1'-oxo-2'-D-hydroxypropyl-tetrahydropterin in Dictyostelium discoideum Ax2.

The biosynthesis of D-threo-tetrahydrobiopterin (DH4, tetrahydrodictyopterin) in Dictyostelium discoideum Ax2 was investigated through the mutant disrupted in the gene encoding sepiapterin reductase (SR) by insertional inactivation. The mutant cells, being completely devoid of SR protein, showed 18.1% of L-erythro-tetrahydrobiopterin (BH4) and 0.6% of DH4 productions in the wild type cells. The mutant cells were also identified to excrete D- and L-sepiapterin, which were presumed to originate from intracellular 1'-oxo-2'-D-hydroxypropyl- and 1'-oxo-2'-L-hydroxypropyl-tetrahydropterin (H4-pterin), respectively. Furthermore, in a coupled assay with Dictyostelium SR, the mutant cell extract exhibited a novel enzyme activity converting 6-pyruvoyltetrahydropterin to 1'-oxo-2'-D-hydroxypropyl-H4-pterin. These results are clear demonstration of the in vivo synthesis of DH4 via 1'-oxo-2'-D-hydroxypropyl-H4-pterin as well as an alternative synthesis of BH4 and DH4 in the complete absence of SR.

Sepiapterin reductases from Chlorobium tepidum and Chlorobium limicola catalyze the synthesis of L-threo-tetrahydrobiopterin from 6-pyruvoyltetrahydropterin.

The ORF sequences of the gene encoding sepiapterin reductase were cloned from the genomic DNAs of Chlorobium tepidum and Chlorobium limicola, which are known to produce L-threo- and L-erythro-tetrahydrobiopterin (BH4)-N-acetylglucosamine, respectively. The deduced amino acid sequence of C. limicola consists of 241 residues, while C. tepidum SR has three residues more at the C-terminal. The overall protein sequence identity was 87.7%. Both recombinant proteins generated from Escherichia coli were identified to catalyze reduction of diketo compound 6-pyruvoyltetrahydropterin to L-threo-BH4. This result suggests that C. limicola needs an additional enzyme for L-erythro-BH4 synthesis to yield its glycoside. The catalytic activity of Chlorobium SRs also supports the previously proposed mechanism of two consecutive reductions of C1' carbonyl group of 6-pyruvoyltetrahydropterin via isomerization reaction.