Nrf2 regulates the expression of γ-glutamylcysteine synthetase in the inflammatory cells of bronchial asthma guinea pig

AIM:To investigate Nrf2 that regulates the expression of γ-glutamylcysteine synthetase (γ-GCS) in the inflammatory cells in bronchial asthma guinea pig bronchoalveolar lavage fluid (BALF). METHODS:Adult male guinea pigs were randomly divided into control group (group A), asthmatic group (group B) and dexamethasone group (group C). Asthmatic model was established by the method of ovalbumin challenge. MDA concentration in the lung tissue homogenate was detected. The total cell count and the proportion of inflammatory cells in BALF were measured. The methods of immunohistochemistry and in situ hybridization were used for detection of the protein and mRNA expressions of Nrf2, Bach1 and γ-GCS. RESULTS:(1) The proportion of eosinophils (EOS) in BALF and the MDA concentration of the lung tissue in group B were higher than those in group A and group C. (2) The result of in situ hybridization indicated that the A value of γ-GCS was the highest in group A compared to group B and group C, but the A value of Nrf2 and Bach1 in 3 groups has no statistical significance. (3) Immunohistochemistry indicated that the A value of γ-GCS in group B was lower than that in group A. The positive rate in cell nucleus of Nrf2 in group B was lower than that in group A and group C. The positive rate in cell nucleus of Bach1 in group B was higher than that in group A and group C. (4) The mRNA expression of γ-GCS (A value) showed positive correlation with the positive rate in cell nucleus of Nrf2 and negative correlation with the positive rate in cell nucleus. A negative correlation between the proportion of EOS in BALF of group B and the γ-GCS mRNA was observed. CONCLUSION:There is disequilibrium between oxidation and anti-oxidation in bronchial asthma guinea pig. Inflammatory reaction decreases the expression of γ-GCS in the inflammatory cells in bronchial asthma guinea pig. Dexamethasone regulates the nuclear translocation of Nrf2/Bach1 and increases the expression of γ-GCS.     

Effect of erythromycin on transforming growth factor-β1 and γ-glutamylcysteine synthetase in lung of smoking rats

AIM: To investigate the effect of erythromycin on the level of transforming growth factor-β₁ (TGF-β₁) and γ-glutaglutamylcysteine synthetase (γ-GCS) in smoking rats,and to explore the antioxidate therapeutic role of erythromycin in chronic obstructive pulmonary disease.METHODS: Wistar rats were exposed to cigarettes smoking to establish the model.After passive smoking for 4 weeks,erythromycin intragastric intervention was administered continuously for 8 weeks.The expiratory airway resistance and lung compliance were assessed and the expression levels of TGF-β₁ and γ-GCS proteins (and the mRNA) in airway endothelial cells and alveolar macrophages were observed respectively by immunohistochemical,immunocytochemical and (in situ) hybridization.RESULTS: The expiratory airway resistance was increased and the lung compliance was degraded significantly in smoking group and erythromycin group,compared to control group.In erythromycin group,the airway resistance was lower and the lung compliance was higher than that in smoking group (P<0.05).The levels of TGF-β₁ and γ-GCS in smoking group and erythromycin group was obviously increased in airway endothelial cells and alveolar macrophages in comparison with control group (P<0.05).The levels of TGF-β₁ and γ-GCS were inhibited by erythromycin (P<0.05).TGF-β₁ was obviously positive correlated with γ-GCS in smoking group,but this was not found in erythromycin group.CONCLUSION: Erythromycin therapy improves pulmonary function and relieves emphysema change induced by smoking in rats,and decreases the expression of TGF-β₁ and γ-GCS in alveolar macrophages and airway endothelial cells,suggesting that erythromycin may play a role in the antioxidate therapeutic in COPD.     

Development of hypo-allergenic apples: silencing of the major allergen Mal d 1 gene in ‘Elstar’ apple and the effect of grafting

Summary

Many people who are allergic to birch pollen are also allergic to apple fruit, due to cross- allergenicity. Since apples are the most extensively consumed fruit in Europe, it is highly relevant to develop a hypo-allergenic apple.Apples with significantly reduced levels of the allergen, Mal d 1, may allow many apple allergics to eat them without an allergic reaction. We are currently collaborating to develop a hypo-allergenic apple within the European Integrated Research Project, ISAFRUIT (www.isafruit.org). Hypo-allergenic apple plants (Malus × domestica Borkh., ‘Elstar’) with decreased levels of Mal d 1 mRNA were produced by RNA interference (RNAi) technology. Ten genetically modified (GM) apple lines were selected. In vitro plantlets were first transferred to a greenhouse, then grafted onto wild-type M.9 rootstock to promote the development of fruit-producing trees. Levels of Mal d 1 gene silencing were measured repeatedly by quantitative real-time PCR. Compared to leaf samples from wild-type ‘Elstar’, two GM lines showed modest levels of gene silencing (up to 250-fold), whereas the other eight GM lines were significantly silenced (up to10,000-fold) in Mal d 1 gene expression. These levels of silencing were unaffected by grafting, and have been stable over more than 3 years, and throughout all developmental stages.     

Evaluation of the sensitivity to ionising γ-radiation of a Cymbidium hybrid

Gamma-rays are an important mutagenic agent that can induce new, useful genetic variations in plants. However, γ-irradiation can also cause damage that negatively affects the use of such mutagens in plant breeding. The aim of the present study was to investigate the effects and damage caused by γ-radiation in a Cymbidium hybrid, RB001. The relative growth rate of protocorm-like bodies (PLBs) was reduced by 50% at a γ-ray dose of approximately 40 Gy. Malondialdehyde concentrations increased significantly with increasing radiation dose. However, almost no difference was observed between untreated control PLBs and PLBs treated with 200 Gy 8 weeks after γ-irradiation. The activities of several antioxidant defence enzymes increased gradually with increasing γ-ray dose, 24 h after irradiation. These enzymes showed different responses between 1 and 4 weeks, but no difference 8 weeks after irradiation. The ‘comet assay’ and flow cytometry were performed. Clear differences in radiation-induced damage were observed between control and 200 Gy-treated PLBs at 24 h. However, PLBs had a tendency to recover from 4 weeks after irradiation, and the integrity of their DNA was similar in samples treated with 10–200 Gy. These results indicated that γ-rays caused little DNA damage and the plants could recover. This demonstrated the feasibility of using physiological responses, the ‘comet assay’, and flow cytometry to detect DNA damage after γ-irradiation.     

Sequence analysis,prokaryotic expression and antiserum preparation of coat protein gene of Actinidia virus A in Sichuan province北大核心CSCD

【Objective】The purpose of the research was to study the occurrence and molecular diversity of Actinidia virus A (AcVA), so as to provide scientific basis for the rapid detection, scientific prevention and control of AcVA and the variety breeding of kiwifruit in China.【Methods】Total RNA was extracted from kiwifruit leaves by CTAB (Cetyl Trimethyl Ammonium Bromide). Nested- PCR was used to amplify AcVA CP gene sequence. The first round PCR amplification was performed by using primers AcVA- 1F (5’- ATGAATCGTTCGAGCA TAGGT- 3’) and AcVA- 1R (5’- TGCGAACATGGTCCCACACTTA-3’), and the pair of primers was designed according to the full length of AcVA sequence, and the amplified fragment was 888 bp that included the complete CP gene sequence. The reaction was conducted under conditions of initial 5 min denaturation at 94 ℃, 34 cycles of 94 ℃ for 60 s, 54 ℃ for 60 s, 72 ℃ for 60 s and extension for 10 min at 72 ℃. Then, 1 μL PCR product was used as template for the second round of PCR amplification with the primers AcVA- 2F (5’- ATGGCAAAGAATATCTCAAG-3’) and AcVA-2R (5’-CTATATTTCAACAGCCTGC-3’). PCR was performed using the following parameters: one cycle at 94 ℃ for 5 min, 34 cycles at 94 ℃ for 30 s, 54 ℃ for 30 s, and 72 ℃ for 40 s, and extension for 10 min at 72 ℃. The BLAST algorithm was used to search the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) databases for homologous sequences and ascertain the identity of target gene. DNAMAN was used to analyze the AcVA CP gene sequence, and MegAlign was used to analyze the sequence identity. The phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA 6.0. The restriction enzymes Bam HⅠ and Sal Ⅰ were added to the corresponding end of the AcVA CP gene sequence, respectively. The PCR purified products and pET28a vector were digested by Bam HⅠ and Sal Ⅰ, after that they were ligated with T4 DNA ligase (TaKaRa) and transferred into E. coli (Escherichia coli) strains DH5á (TaKaRa), and finally plated onto LB (Luria-Bertani) agar containing Kana (Kanamycin). The expression strain BL21 containing the recombinant plasmid was cultured at 37 ℃ overnight, and transferred to a new medium at a 10% inoculum on the second day, until OD600 reached 0.4-0.6, IPTG (Isopropyl β-D-Thiogalactoside) was added to a final concentration of 0.2, 0.4, 0.6, 0.8, 1.0 mM, and incubated at 16 ℃ overnight. The expressed protein was purified and then anti-AcVA-CP antibody was obtained from a rabbit. The optimal titer of the antiserum was tested by Western blot.【Results】The complete CP gene sequences of AcVA Sichuan isolates were cloned by nested-PCR and named as AcVA-DJY4, AcVA-HYZ1 and AcVA-WBS12, respectively. In the study the frequency of AcVA in Sichuan province was also counted. The full length of the CP gene sequence was 597 bp, encoding 198 amino acids. Based on the comparison of the nucleotide sequence and the deduced amino acid sequence of CP gene of the AcVA isolates, the nucleotide identity between the AcVA isolates from Sichuan province ranged from 86.9% to 88.3%, and the identity of the encoded amino acids ranged from 96.0 to 96.5%. The CP genes of three AcVA Sichuan isolates shared 87.9%-90.8% nucleotide identity and 94.9%-97.5% amino acid identity with the Actinidia virus A isolate TP7-93A (AcVA-TP7-93A) from New Zealand and the Actinidia virus A isolate Haenam (AcVA-Haenam) from South Korea. Phylogenetic analysis based on nucleotide sequence of AcVA CP gene showed that AcVA-HYZ1 was clustered with AcVA-Haenam (Group I), and AcVA-WBS12 was clustered with AcVA-TP7-93A (Group II), but AcVA-DJY4 was an independent branch. Phylogenetic analysis based on amino acid sequence of AcVA CP gene showed that AcVA-WBS12 was a single branch, AcVA-DJY4 and AcVA-HYZ1 were clustered into one group (Group I), and the AcVA-Haenam reported in South Korea and the AcVA-TP7-93A reported in New Zealand were clustered into another group (Group II). The results of the phylogenetic analyses indicated that there were significant differences among the AcVA isolates. The prokaryotic expression plasmid pET28a-AcVA-DJY4-CP was successfully constructed. The target fusion protein (27 kDa) was highly expressed in E. coli induced by 0.6 mmol · L- 1 IPTG at 16 ℃. The expressed protein was purified and retrieved and then used to immune rabbits and the corresponding specific antiserum was prepared. Western blot analysis confirmed that the antiserum reacted strongly and specifically with the CP of AcVA-DJY4, with the optimal titer of the antiserum being up to 1:5 000.【Conclusion】Three AcVA Sichuan isolates were obtained for the first time, enriching the sequence diversity of the AcVA CP gene sequences. In the study, the optimal conditions were explored for prokaryotic expression and specific antisera was prepared for detection of AcVA-DJY4-CP. Western blot analysis showed that the antiserum could be used for the detection of AcVA in the kiwifruit producing districts. These results can also provide a technical support for the rapid detection, scientific prevention and control of virus disease in kiwifruit plant and the breeding of kiwifruit varieties. © 2019 Journal of Fruit Science     

Green roofs in temperate climate cities in Europe – An analysis of key decision factors

This paper aims to identify and assess the main decision factors that are relevant for the diffusion of green roof technology in cities of temperate climate in Europe. A mixed design method was applied to identify relevant factors and to structure these along a Strengths, Weaknesses, Opportunities and Threats framework. The factors were subsequently assessed by a sample of green roof experts, using an Analytical Hierarchy Process. The results indicate that prospects for green roofs are in general rather bright, and that dissemination potential is substantial. Green roofs are particularly likely to benefit from climate change and respective counter-strategies, as they are seen as an adaptation and mitigation measure. However, current barriers to adoption need to be carefully considered. Especially the dilemmatic incentive structures, in that building owners bear most of the risks and potential disadvantages, while the public collectively benefits from green roof advantages, could be a major implementation barrier. Without supportive policies, green roofs are thus unlikely to move from niche to regime level in the near future.     

Woody species composition of chestnut stands in the Northern Apennines: the result of 200 years of changes in land use

Chestnut stands (orchards and coppices) are among the most typical elements of the southern European mountain landscape and a protected habitat (9260 Castanea sativa woods) according to the European Union (Directive 92/43/EEC). As an anthropogenic landscape, they require specific measures to address preservation or to guide their evolutionary trend. In the Northern Apennines, a landscape multiscalar-multitemporal approach was adopted to highlight factors that have acted on the evolution of this habitat and which still might affect either its preservation or its evolutionary dynamics. Using a diachronic GIS-approach, we analyzed old cadastral maps (drawn up 200 years ago), and aerial photographs. Both the present distribution pattern of the woody species and the incidence of important chestnut diseases were also surveyed. The factors explaining the current extent and species composition of the local chestnut forests confirm their status as an anthropogenic habitat. The present landscape distribution of chestnut woods is heavily linked to past human settlements. Chestnut blight and ink disease are more an indirect reason for past felling activities than an actual direct cause of damage to trees, because of the hypovirulence spread and the limited incidence of the ink disease. Vegetation dynamics of abandoned chestnut forests evolved only partly towards deciduous Beech and Hop Hornbeam stands, thus suggesting both the possibility of a recovery of this cultivation and the need for new criteria for its management.     

Preliminary trials to examine the effects of ethephon as a thinner of ‘Gala’ and ‘Jonagold’ apples

Trials in southern Tasmania examined the thinning effects of ethephon on ‘Gala’ and ‘Jonagold’ apples. Untreated controls were compared with ethephon thinning sprays applied at full bloom at 50,100,200,400,800 and 1 600 mg I“1 to both cultivars. Thinning of both cultivars was related to the concentration of the spray and in most cases logistic models were fitted. Thinning effectiveness was largely reflected in increased fruit weight and size. The ‘Gala’ fruit was still not large enough for Australian markets. Ethephon at 200 mg I“1 effectively thinned ‘Jonagold’ and produced the required increases in fruit size and weight. ‘Jonagold’ was overthinned by the high concentrations of ethephon, but this was not reflected in increased fruit weight and size. Ethephon also reduced vegetative growth at the higher concentrations which was considered an advantage. More work is required to establish specific recommendations for either cultivar.     

Effects of phosphorylation-defective retinoic acid receptor α1 on proli-feration of human multiple myeloma cells in vitro

AIM: To investigate the effect of phosphorylation-defective retinoic acid receptor α1 (RARα1) on the proliferation of human multiple myeloma cells. METHODS: The mRNA expression of RARα subtypes in U266 cells was detected by RT-PCR. Lentiviral plasmid construction, viral production, titer determination and cell transfection were carried out by the general methods of molecular biology. Proliferation analysis was performed with CCK-8 assay. The U266 cells were treated with all-trans retinoic acid (ATRA,0~100 μmol/L) or transfected with lentivirus RARαS77A. The expression levels of proliferation-related proteins, P53 and Rb, in U266 cells treated with ATRA or transfected with lentivirus RARαS77A were detected by Western blotting. RESULTS: RARα1 was positively expressed in U266 cells and RARα2 expression was negative. ATRA significantly inhibited the proliferation of U266 cells in a dose- and time-dependent manner. Proliferation of U266 cells was significantly inhibited 48 h after transfection with lentivirus RARαS77A, and the inhibitory rate was 15.16%±3.84%. The up-regulated expression of Rb and down-regulated expression of P53 were detected in U266 cells not only in the cells treated with ATRA, but also in the cells transfected with lentivirus RARαS77A. CONCLUSION: Phosphorylation-defective RARα1 (RARαS77A) mimics the growth inhibitory effect of ATRA on U266 cells that express RARα1 (+) and RARα2 (-) via down-regulating the expression of P53 and up-regulating the expression of Rb, suggesting that the antiproliferative effect of ATRA is mainly mediated by decreasing the phosphorylation of RARα1.     

Expression of PPARγ, Nrf2 and γ-GCS-h in inflammatory cells in bronchoalveolar lavage fluid of guinea pigs with bronchial asthma

AIM: To investigate the expression of PPARγ and Nrf2/γ-GCS-h in inflammatory cells in bronchoalveolar lavage fluid(BALF) of guinea pig with bronchial asthma of acute episode, and to explore the roles of PPARγ on Nrf2/γ-GCS-h expression. METHODS: Forty adult male guinea pigs were randomly divided into 4 groups (10 guinea pigs in each group): control group (group A), asthmatic group (group B), dexamethasone treatment group (group C) and rogridone treatment group (group D). The asthmatic model was established by an ovalbumin challenge method. BALF was collected, and the total cell count and the proportion of the inflammatory cells were measured. After centrifugation, the concentrations of ROS and MDA in the clear supernatant were detected. The methods of in situ hybridization and immunohistochemistry were used for detecting the expression of PPARγ and Nrf2/γ-GCS-h at mRNA and protein levels. RESULTS: The proportion of eosinophils (EOS) in BALF in group B was significantly higher than that in groups A, C and D (P<0.01). The concentrations of ROS and MDA in BALF of group B was the highest. The difference of ROS and MDA was statistically significant (all P<0.05) as compared to the control. The results of immunohistochemistry and in situ hybridization indicated that the A value was the lowest in group B as compared to that in groups A, C and D (all P<0.01). In group B, the positive correlations were observed between PPARγ and Nrf2/γ-GCS-h, between γ-GCS-h and Nrf2. A negative correlation between the proportion of EOS in BALF and the expression of PPARγ and Nrf2/γ-GCS-h was also observed (all P<0.05). CONCLUSION: In acute asthmatic models induced by ovalbumin, the expression of PPARγ and Nrf2/γ-GCS-h is decreased, and PPARγ may up-regulate the expression of Nrf2/γ-GCS-h to inhibit the inflammatory and oxidative reactions, indicating a new way for prevention and treatment of bronchial asthma.     

Socioecological features of plant diversity in domestic gardens in the city of Bogotá, Colombia

Domestic gardens contribute substantially to green spaces in cities, where urbanization removes and fragments natural landscapes, causing the loss of biodiversity and the homogenization of biota. We analyzed the diversity and composition of the flora of 70 domestic gardens in seven localities in Bogotá, Colombia, which represent different periods of expansion of the city. Floristic composition and diversity were related to the origin and use of plants, the urbanization history, and the income of owners. We recorded 4110 individuals belonging to 238 species. The mean species richness per garden was 15.4, with older localities having significantly higher species richness. The similarity among the localities, which evaluated the distinctness of assemblages, ranged from 0.42 to 0.50. Plants from the Neotropical region and exotic plants were the most abundant in all gardens. The most common use was ornamental, and use depended on the socioeconomic status of the owner. The lower-income homes cultivated larger proportions of edible and medicinal plants. Gardens at the oldest localities, with the largest number of native species, contribute to the conservation of flora because they contain the largest number of native species. Furthermore, domestic gardens are good sources of employment for gardeners and are useful places to keep senior citizens active, helping these citizens relieve stress, maintain good health and teach young people the cultural uses of plants. The receptivity of the homeowners to the study opens the door to future research and conservation programs in the city.