The transformation of banana with potential virus resistance genes

One approach to reducing the yield losses caused by banana viral diseases is the use of genetic engineering and pathogen-derived resistance strategies to generate resistant cultivars. The development of transgenic virus resistance requires an efficient banana transformation method, particularly for commercially important 'Cavendish' type cultivars such as 'Grand Nain'. Prior to this study, only two examples of the stable transformation of banana had been reported, both of which demonstrated the principle of transformation but did not characterise transgenic plants in terms of the efficiency at which individual transgenic lines were generated, relative activities of promoters in stably transformed plants, and the stability of transgene expression. The aim of this study was to develop more efficient transformation methods for banana, assess the activity of some commonly used and also novel promoters in stably transformed plants, and transform banana with genes that could potentially confer resistance to banana bunchy top nanovirus (BBTV) and banana bract mosaic potyvirus (BBrMV). A regeneration system using immature male flowers as the explant was established. The frequency of somatic embryogenesis in male flower explants was influenced by the season in which the inflorescences were harvested. Further, the media requirements of various banana cultivars in respect to the 2,4-D concentration in the initiation media also differed. Following the optimisation of these and other parameters, embryogenic cell suspensions of several banana (Musa spp.) cultivars including 'Grand Nain' (AAA), 'Williams' (AAA), 'SH-3362' (AA), 'Goldfinger' (AAAB) and 'Bluggoe' (ABB) were successfully generated. Highly efficient transformation methods were developed for both 'Bluggoe' and 'Grand Nain'; this is the first report of microprojectile bombardment transformation of the commercially important 'Grand Nain' cultivar. Following bombardment of embryogenic suspension cells, regeneration was monitored from single transfom1ed cells to whole plants using a reporter gene encoding the green fluorescent protein (gfp). Selection with kanamycin enabled the regeneration of a greater number of plants than with geneticin, while still preventing the regeneration of non-transformed plants. Southern hybridisation confirmed the neomycin phosphotransferase gene (npt II) was stably integrated into the banana genome and that multiple transgenic lines were derived from single bombardments. The activity, stability and tissue specificity of the cauliflower mosaic virus 358 (CaMV 35S) and maize polyubiquitin-1 (Ubi-1) promoters were examined. In stably transformed banana, the Ubi-1 promoter provided approximately six-fold higher p-glucuronidase (GUS) activity than the CaMV 35S promoter, and both promoters remained active in glasshouse grown plants for the six months they were observed. The intergenic regions ofBBTV DNA-I to -6 were isolated and fused to either the uidA (GUS) or gfjJ reporter genes to assess their promoter activities. BBTV promoter activity was detected in banana embryogenic cells using the gfp reporter gene. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that generated by the maize Ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter (BT6.1) was restricted to the phloem of leaves and roots, stomata and root meristems. The activity of the BT6.1 promoter was enhanced by the inclusion of intron-containing fragments derived from the maize Ubi-1, rice Act-1, and sugarcane rbcS 5' untranslated regions in GUS reporter gene constructs. In transient assays in banana, the rice Act-1 and maize Ubi-1 introns provided the most significant enhancement, increasing expression levels 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about 10-fold. In stably transformed banana plants, the maize Ubi-1 intron enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35S promoter, but did not appear to alter the tissue specificity of the promoter. Both 'Grand Nain' and 'Bluggoe' were transformed with constructs that could potentially confer resistance to BBTV and BBrMV, including constructs containing BBTV DNA-1 major and internal genes, BBTV DNA-5 gene, and the BBrMV coat protein-coding region all under the control of the Ubi-1 promoter, while the BT6 promoter was used to drive the npt II selectable marker gene. At least 30 transgenic lines containing each construct were identified and replicates of each line are currently being generated by micropropagation in preparation for virus challenge.

The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB

Examination of relationships between urban form, household activities, and time allocation in the Atlanta Metropolitan Region

A substantial body of research is focused on understanding the relationships between socio-demographics, land-use characteristics, and mode specific attributes on travel mode choice and time-use patterns. Residential and commercial densities, inter-mixing of land uses, and route directness in conjunction with transportation performance characteristics interact to influence accessibility to destinations as well as time spent traveling and engaging in activities. This study uniquely examines the activity durations undertaken for out-of-home subsistence; maintenance, and discretionary activities. Also examined are total tour durations (summing all activity categories within a tour). Cross-sectional activities are obtained from household activity travel survey data from the Atlanta Metropolitan Region. Time durations allocated to weekdays and weekends are compared. The censoring and endogeneity between activity categories and within individuals are captured using multiple equations Tobit models. The analysis and modeling reveal that land-use characteristics such as net residential density and the number of commercial parcels within a kilometer of a residence are associated with differences in weekday and weekend time-use allocations. Household type and structure are significant predictors across the three activity categories, but not for overall travel times. Tour characteristics such as time-of-day and primary travel mode of the tours also affect traveler's out-of-home activity-tour time-use patterns.

Cigarette smoking in young adults : the influence of the HTR2A T102C polymorphism and punishment sensitivity

Background: The C allele of a common polymorphism of the serotonin 2A receptor (HTR2A) gene, T102C, results in reduced synthesis of 5-HT2A receptors and has been associated with current smoking status in adults. The -1438A/G polymorphism, located in the regulatory region of this gene, is in linkage disequilibrium with T102C, and the A allele is associated with increased promoter activity and with smoking in adult males. We investigated the contributions of the HTR2A gene, chronic psychological stress, and impulsivity to the prediction of cigarette smoking status and dependence in young adults. Methods: T102C and -1438A/G genotyping was conducted on 132 healthy Caucasian young adults (47 smokers) who completed self-report measures of chronic stress, depressive symptoms, impulsive personality and cigarette use. Results: A logistic regression analysis of current cigarette smoker user status, after adjusting for gender, depressive symptom severity and chronic stress, indicated that the T102C TT genotype relative to the CC genotype (OR = 7.53), and lower punishment sensitivity (OR = 0.91) were each significant predictive risk factors. However, for number of cigarettes smoked, only lower punishment sensitivity was a significant predictor (OR = 0.81). Conclusions: These data indicate the importance of the T102C polymorphism to tobacco use but not number of cigarettes smoked for Caucasian young adults. Future studies should examine whether this is explained by effects of nicotine on the serotonin system. Lower punishment sensitivity increased risk of both smoking and of greater consumption, perhaps via a reduced sensitivity to cigarette health warnings and negative physiological effects.

The comparison of international and local sustainable assessment tools of landscape design for housing estate developments : case of Bangkok Metropolitan Region, Thailand

Various countries have been introducing sustainable assessment tools for real estate design to produce integrated sustainability components not just for the building, but also the landscape component of the development. This paper aims to present the comparison between international and local assessment tools of landscape design for housing estate developments in Bangkok Metropolitan Region (BMR), Thailand. The methodologies used are to review, then compare and identify discrepancy indicators among the tools. This paper will examine four international tools; LEED for Neighbourhood Development (LEED – ND) of United State of America (USA), EnviroDevelopment standards of Australia, Residential Landscape Sustainability of United Kingdom (UK) and Green Mark for Infrastructure of Singapore; and three BMR’s existing tools; Land Subdivision Act B.E. 2543, Environmental Impact Assessment Monitoring Awards (EIA-MA) and Thai’s Rating for Energy and Environmental Sustainability of New construction and major renovation (TREES-NC). The findings show that there are twenty two elements of three categories which are neighbourhood design, community management, and environmental condition. Moreover, only one element in neighbourhood designs different between the international and local tools. The sustainable assessment tools have existed in BMR but they are not complete in only one assessment tool. Thus, the development of new comprehensive assessment tool will be necessary in BMR; however, it should meet the specific environment and climate condition for housing estate development at BMR.