De novo sequence analysis of cytochrome P450 1–3 genes expressed in ostrich liver with highest expression of CYP2G19

The cytochrome P450 (CYP) 1–3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1–3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2–10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism.     

A passive integrated transponder tag implanted by a new alternative surgical method: effects on the oriental weather loach (Misgurnus anguillicaudatus) and application in a small irrigation system

We validated the effects of a passive integrated transponder (PIT) tagging process on the oriental weather loach Misgurnus anguillicaudatus. Laboratory experiments were conducted to assess the effects of PIT tagging on fish survival, growth, wound healing, and tag omission. Two tagging protocols, standard syringe injection versus insertion through a small hole pierced by a fine needle-shaped awl, were compared using a 12.5 × 2.07 mm² tag. A control group was also included. In comparison with the awl technique, syringe injection heightened the mortality of the loach and delayed healing of the wound caused by tag insertion. No effects of either PIT tagging method were detected on the growth of surviving loach. We also field-tested similarly tagged populations within a river-based irrigation system of Sado Island, Japan. Two different sized tags (long, 12.5 × 2.07 mm²; short, 8.5 × 2.12 mm²) were compared by using antenna loggers which detected fish movement through gates and automatically logged tagged fish’s tag IDs and timestamps. By comparing logged data and actual fish collection surveys both below and above the gates, 77% and 30% of actual loach movements were confirmed to have been successfully logged for the long and short tags, respectively. The awl insertion technique with the longer tag is therefore recommended for use in similar studies of smaller fish species.     

<Emphasis Type="Italic">Bruguiera hainesii</Emphasis>, a critically endangered mangrove species,is a hybrid between <Emphasis Type="Italic">B. cylindrica</Emphasis> and <Emphasis Type="Italic">B. gymnorhiza</Emphasis> (Rhizophoraceae)

Bruguiera hainesii (Rhizophoraceae) is one of the two Critically Endangered mangrove species listed in the IUCN Red List of Threatened Species. Although the species is vulnerable to extinction, its genetic diversity and the evolutionary relationships with other Bruguiera species are not well understood. Also, intermediate morphological characters imply that the species might be of hybrid origin. To clarify the genetic relationship between B. hainesii and other Bruguiera species, we conducted molecular analyses including all six Bruguiera species using DNA sequences of two nuclear genes (CesA and UNK) and three chloroplast regions (intergenic spacer regions of trnL-trnF, trnS-trnG and atpB-rbcL). For nuclear DNA markers, all nine B. hainesii samples from five populations were heterozygous at both loci, with one allele was shared with B. cylindrica, and the other with B. gymnorhiza. For chloroplast DNA markers, the two haplotypes found in B. hainesii were shared only by B. cylindrica. These results suggested that B. hainesii is a hybrid between B. cylindrica as the maternal parent and B. gymnorhiza as the paternal one. Furthermore, chloroplast DNA haplotypes found in B. hainesii suggest that hybridization has occurred independently in regions where the distribution ranges of the parental species meet. As the IUCN Red List of Threatened Species currently excludes hybrids (except for apomictic plant hybrids), the conservation status of B. hainesii should be reconsidered.     

Intensive determination of storage condition effects on human plasma metabolomics

Introduction

Human plasma metabolomics offer powerful tools for understanding disease mechanisms and identifying clinical biomarkers for diagnosis, efficacy prediction and patient stratification. Although storage conditions can affect the reliability of data from metabolites, strict control of these conditions remains challenging, particularly when clinical samples are included from multiple centers. Therefore, it is necessary to consider stability profiles of each analyte.

Objectives

The purpose of this study was to extract unstable metabolites from vast metabolome data and identify factors that cause instability.

Method

Plasma samples were obtained from five healthy volunteers, were stored under ten different conditions of time and temperature and were quantified using leading-edge metabolomics. Instability was evaluated by comparing quantitation values under each storage condition with those obtained after ?80 °C storage.

Result

Stability profiling of the 992 metabolites showed time- and temperature-dependent increases in numbers of significantly changed metabolites. This large volume of data enabled comparisons of unstable metabolites with their related molecules and allowed identification of causative factors, including compound-specific enzymatic activity in plasma and chemical reactivity. Furthermore, these analyses indicated extreme instability of 1-docosahexaenoylglycerol, 1-arachidonoylglycerophosphate, cystine, cysteine and N⁶-methyladenosine.

Conclusion

A large volume of data regarding storage stability was obtained. These data are a contribution to the discovery of biomarker candidates without misselection based on unreliable values and to the establishment of suitable handling procedures for targeted biomarker quantification.

     

A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

Mitochondrial oxidative stress is considered as a key accelerator of fibrosis in various organs including the liver. However, the production of oxidative stress and progression of liver fibrosis may merely represent the independent consequences of hepatocellular injury caused by the primary disease. Because of a lack of appropriate experimental models to evaluate the sole effects of oxidative stress, it is virtually unknown whether this stress is causatively linked to the progression of liver fibrosis. Here, we examined the direct effects of mitochondrial reactive oxygen species (ROS) on the progression of high fat/calorie diet-induced steatohepatitis using Tet-mev-1 mice, in which a mutated succinate dehydrogenase transgene impairs the mitochondrial electron transport and generates an excess amount of ROS in response to doxycycline administration. Wild type and Tet-mev-1 mice that had been continuously given doxycycline-containing water were subsequently fed either normal chow or a cholesterol-free high-fat/high-sucrose diet for 4 months at approximately 1 or 2 years of age. Histopathological examinations indicated that neither the mitochondrial ROS induced in Tet-mev-1 mice nor the feeding of wild type animals with high-fat/high-sucrose diet alone caused significant liver fibrosis. Only when the Tet-mev-1 mice were fed a high-fat/high-sucrose diet, it induced lipid peroxidation in hepatocytes and enhanced hepatic CC chemokine expression. These events were accompanied by increased infiltration of CCR5-positive cells and activation of myofibroblasts, resulting in extensive liver fibrosis. Interestingly, this combinatorial effect of mitochondrial ROS and excess fat/calorie intake on liver fibrosis was observed only in 2-year-old Tet-mev-1 mice, not in the 1-year-old animals. Collectively, these results indicate that mitochondrial ROS in combination with excess fat/calorie intake accelerates liver fibrosis by enhancing CC chemokine production in aged animals. We have provided a good experimental model to explore how high fat/calorie intake increases the susceptibility to nonalcoholic steatohepatitis in aged individuals who have impaired mitochondrial adaptation.     

Clinical Course and Changes in High-Resolution Computed Tomography Findings in Patients with Idiopathic Pulmonary Fibrosis without Honeycombing

Some patients with idiopathic pulmonary fibrosis (IPF) do not have honeycombing on high-resolution computed tomography (HRCT) at their initial evaluation. The clinical course and sequential changes in HRCT findings in these patients are not fully understood. We reviewed the cases of 43 patients with IPF without honeycombing on initial HRCT from institutions throughout Japan. All patients were diagnosed with IPF based on a surgical lung biopsy. Multidisciplinary discussions were held five times between 2011 and 2014, to exclude alternative etiologies. We evaluated the sequential changes in HRCT findings in 30 patients with IPF. We classified these 30 patients into three groups based on their HRCT patterns and clarified the clinical characteristics and prognosis among the groups. The patterns of all 30 patients on initial HRCT corresponded to a possible usual interstitial pneumonia (UIP) pattern which was described in the 2011 International Statement. On long-term follow-up (71.0±38.7 standard deviation [SD] months), honeycombing was seen in 16 patients (53%, the HoneyCo group); traction bronchiectasis or cysts without honeycombing was observed in 12 patients (40%, the NoHoneyCo group), and two patients showed no interval change (7%, the NoChange group) on HRCT. The mean survival periods of the HoneyCo and NoHoneyCo groups were 67.1 and 61.2 months, respectively (p = 0.76). There are some patients with IPF whose conditions chronically progress without honeycombing on HRCT. The appearance of honeycombing on HRCT during the follow-up might not be related to prognosis.     

Association between Smoking Status and Obesity in a Nationwide Survey of Japanese Adults

Objective

A positive association between the number of cigarettes smoked per day and obesity has been reported, whereas how other smoking-related indices, such as pack-years and duration of smoking, are related with obesity has been less investigated. We analyzed the age-adjusted cross-sectional association between smoking and obesity in a general Japanese population.

Methods

We used data from a nationwide epidemiological study of Japanese adults (N = 23,106). We compared the prevalence of obesity (defined as body mass index ≥ 25kg/m²) among groups classified by smoking behavior, pack-years, number of cigarettes per day, duration of smoking, and duration and time of smoking cessation.

Results

In men, current smokers had a lower odds ratio (OR) for obesity of 0.80 (95% confidence interval (CI): 0.72–0.88) compared to non-smokers, whereas past smokers had a higher OR of 1.23 (95% CI: 1.09–1.37) compared to current smokers. In women, there were no differences in obesity between the three groups classified by smoking behavior. However, in both sexes, the prevalence of obesity tended to increase with pack-years and the number of cigarettes per day, but not with duration of smoking in current and past smokers. Further, in male smokers, the risks for obesity were markedly higher in short-term heavy smokers compared with long-term light smokers, even with the same number of pack-years. Regarding the impact of smoking cessation, female past smokers who quit smoking at an age > 55-years had an elevated OR of 1.60 (95% CI:1.05–2.38) for obesity.

Conclusions

In a general Japanese population, obesity is progressively associated with pack-years and number of cigarettes per day, but not with the duration of smoking. When investigating the association between obesity and cigarette smoking, the daily smoking burden and the duration of smoking require to be independently considered.     

Expression of an extracellular ribonuclease gene increases resistance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.