Molecular characterisation of six badnavirus species associated with leaf streak disease of banana in East Africa

Banana leaf streak disease, caused by several species of Banana streak virus (BSV), is widespread in East Africa. We surveyed for this disease in Uganda and Kenya, and used rolling-circle amplification (RCA) to detect the presence of BSV in banana. Six distinct badnavirus sequences, three from Uganda and three from Kenya, were amplified for which only partial sequences were previously available. The complete genomes were sequenced and characterised. The size and organisation of all six sequences was characteristic of other badnaviruses, including conserved functional domains present in the putative polyprotein encoded by open reading frame (ORF) 3. Based on nucleotide sequence analysis within the reverse transcriptase/ribonuclease H-coding region of open reading frame 3, we propose that these sequences be recognised as six new species and be designated as Banana streak UA virus, Banana streak UI virus, Banana streak UL virus, Banana streak UM virus, Banana streak CA virus and Banana streak IM virus. Using PCR and species-specific primers to test for the presence of integrated sequences, we demonstrated that sequences with high similarity to BSIMV only were present in several banana cultivars which had tested negative for episomal BSV sequences.

A myosin from a higher plant has structural similarities to class V myosins

In plant cells, myosin is believed to be the molecular motor responsible for actin-based motility processes such as cytoplasmic streaming and directed vesicle transport. In an effort to characterize plant myosin, a cDNA encoding a myosin heavy chain was isolated from Arabidopsis thaliana. The predicted product of the MYA1 gene is 173 kDa and is structurally similar to the class V myosins. It is composed of the highly-conserved NH2-terminal "head" domain, a putative calmodulin-binding "neck" domain an alpha-helical coiled-coil domain, and a COOH-terminal domain. Northern blot analysis shows that the Arabidopsis MYA1 gene is expressed in all the major plant tissues (flower, leaf, root, and stem). We suggest that the MYA1 myosin may be involved in a general intracellular transport process in plant cells.

Modelling cell separation during plant organ abscission

A simple mathematical model is presented to describe the cell separation process that plants undertake in order to deliberately shed organs. The focus here is on modelling the production of the enzyme polygalacturonase, which breaks down pectin that provides natural cell-to-cell adhesion in the localised abscission zone. A coupled system of three ordinary differential equations is given for a single cell, and then extended to hold for a layer of cells in the abscission zone. Simple observations are made based on the results of this preliminary model and, furthermore, a number of opportunities for applied mathematicians to make contributions in this subject area are discussed.

The ghrelin receptor isoforms (GHS-R1a and GHS-R1b) and GPR39 : an investigation into receptor dimerisation

Prostate cancer is the second most common cause of cancer-related deaths in Western males. Current diagnostic, prognostic and treatment approaches are not ideal and advanced metastatic prostate cancer is incurable. There is an urgent need for improved adjunctive therapies and markers for this disease. GPCRs are likely to play a significant role in the initiation and progression of prostate cancer. Over the last decade, it has emerged that G protein coupled receptors (GPCRs) are likely to function as homodimers and heterodimers. Heterodimerisation between GPCRs can result in the formation of novel pharmacological receptors with altered functional outcomes, and a number of GPCR heterodimers have been implicated in the pathogenesis of human disease. Importantly, novel GPCR heterodimers represent potential new targets for the development of more specific therapeutic drugs. Ghrelin is a 28 amino acid peptide hormone which has a unique n-octanoic acid post-translational modification. Ghrelin has a number of important physiological roles, including roles in appetite regulation and the stimulation of growth hormone release. The ghrelin receptor is the growth hormone secretagogue receptor type 1a, GHS-R1a, a seven transmembrane domain GPCR, and GHS-R1b is a C-terminally truncated isoform of the ghrelin receptor, consisting of five transmembrane domains. Growing evidence suggests that ghrelin and the ghrelin receptor isoforms, GHS-R1a and GHS-R1b, may have a role in the progression of a number of cancers, including prostate cancer. Previous studies by our research group have shown that the truncated ghrelin receptor isoform, GHS-R1b, is not expressed in normal prostate, however, it is expressed in prostate cancer. The altered expression of this truncated isoform may reflect a difference between a normal and cancerous state. A number of mutant GPCRs have been shown to regulate the function of their corresponding wild-type receptors. Therefore, we investigated the potential role of interactions between GHS-R1a and GHS-R1b, which are co-expressed in prostate cancer and aimed to investigate the function of this potentially new pharmacological receptor. In 2005, obestatin, a 23 amino acid C-terminally amidated peptide derived from preproghrelin was identified and was described as opposing the stimulating effects of ghrelin on appetite and food intake. GPR39, an orphan GPCR which is closely related to the ghrelin receptor, was identified as the endogenous receptor for obestatin. Recently, however, the ability of obestatin to oppose the effects of ghrelin on appetite and food intake has been questioned, and furthermore, it appears that GPR39 may in fact not be the obestatin receptor. The role of GPR39 in the prostate is of interest, however, as it is a zinc receptor. Zinc has a unique role in the biology of the prostate, where it is normally accumulated at high levels, and zinc accumulation is altered in the development of prostate malignancy. Ghrelin and zinc have important roles in prostate cancer and dimerisation of their receptors may have novel roles in malignant prostate cells. The aim of the current study, therefore, was to demonstrate the formation of GHS-R1a/GHS-R1b and GHS-R1a/GPR39 heterodimers and to investigate potential functions of these heterodimers in prostate cancer cell lines. To demonstrate dimerisation we first employed a classical co-immunoprecipitation technique. Using cells co-overexpressing FLAG- and Myc- tagged GHS-R1a, GHS-R1b and GPR39, we were able to co-immunoprecipitate these receptors. Significantly, however, the receptors formed high molecular weight aggregates. A number of questions have been raised over the propensity of GPCRs to aggregate during co-immunoprecipitation as a result of their hydrophobic nature and this may be misinterpreted as receptor dimerisation. As we observed significant receptor aggregation in this study, we used additional methods to confirm the specificity of these putative GPCR interactions. We used two different resonance energy transfer (RET) methods; bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), to investigate interactions between the ghrelin receptor isoforms and GPR39. RET is the transfer of energy from a donor fluorophore to an acceptor fluorophore when they are in close proximity, and RET methods are, therefore, applicable to the observation of specific protein-protein interactions. Extensive studies using the second generation bioluminescence resonance energy transfer (BRET2) technology were performed, however, a number of technical limitations were observed. The substrate used during BRET2 studies, coelenterazine 400a, has a low quantum yield and rapid signal decay. This study highlighted the requirement for the expression of donor and acceptor tagged receptors at high levels so that a BRET ratio can be determined. After performing a number of BRET2 experimental controls, our BRET2 data did not fit the predicted results for a specific interaction between these receptors. The interactions that we observed may in fact represent ‘bystander BRET’ resulting from high levels of expression, forcing the donor and acceptor into close proximity. Our FRET studies employed two different FRET techniques, acceptor photobleaching FRET and sensitised emission FRET measured by flow cytometry. We were unable to observe any significant FRET, or FRET values that were likely to result from specific receptor dimerisation between GHS-R1a, GHS-R1b and GPR39. While we were unable to conclusively demonstrate direct dimerisation between GHS-R1a, GHS-R1b and GPR39 using several methods, our findings do not exclude the possibility that these receptors interact. We aimed to investigate if co-expression of combinations of these receptors had functional effects in prostate cancers cells. It has previously been demonstrated that ghrelin stimulates cell proliferation in prostate cancer cell lines, through ERK1/2 activation, and GPR39 can stimulate ERK1/2 signalling in response to zinc treatments. Additionally, both GHS-R1a and GPR39 display a high level of constitutive signalling and these constitutively active receptors can attenuate apoptosis when overexpressed individually in some cell types. We, therefore, investigated ERK1/2 and AKT signalling and cell survival in prostate cancer the potential modulation of these functions by dimerisation between GHS-R1a, GHS-R1b and GPR39. Expression of these receptors in the PC-3 prostate cancer cell line, either alone or in combination, did not alter constitutive ERK1/2 or AKT signalling, basal apoptosis or tunicamycin-stimulated apoptosis, compared to controls. In summary, the potential interactions between the ghrelin receptor isoforms, GHS-R1a and GHS-R1b, and the related zinc receptor, GPR39, and the potential for functional outcomes in prostate cancer were investigated using a number of independent methods. We did not definitively demonstrate the formation of these dimers using a number of state of the art methods to directly demonstrate receptor-receptor interactions. We investigated a number of potential functions of GPR39 and GHS-R1a in the prostate and did not observe altered function in response to co-expression of these receptors. The technical questions raised by this study highlight the requirement for the application of extensive controls when using current methods for the demonstration of GPCR dimerisation. Similar findings in this field reflect the current controversy surrounding the investigation of GPCR dimerisation. Although GHS-R1a/GHS-R1b or GHS-R1a/GPR39 heterodimerisation was not clearly demonstrated, this study provides a basis for future investigations of these receptors in prostate cancer. Additionally, the results presented in this study and growing evidence in the literature highlight the requirement for an extensive understanding of the experimental method and the performance of a range of controls to avoid the spurious interpretation of data gained from artificial expression systems. The future development of more robust techniques for investigating GPCR dimerisation is clearly required and will enable us to elucidate whether GHS-R1a, GHS-R1b and GPR39 form physiologically relevant dimers.

Sensitivity of Dot-ELISA on Nitrocellulose Membranes in Comparison with ELISA on Polystyrene Plates for the Detection of Four Plant Viruses

ABSTR.4CT Senitivity of dot-immunobindinding ELf SA on nitrocellulose membrane (DotELISA)was compared with double-antibody sandwich ELISA (DAS-ELlSA) on polystyrene plates for the detection of bean yellow mosaic virus (BYMV), broad bean stain virus (WMV-2). Dot-ELISA was 2 and 1O times more sensitive than DAS-ELISA for the detection of BBSV and WMV-2, respectively, whereas DAS-ELISA was more sensitive than Dot-ELiSA for {he detection of BYMV. Both techniques were equally sensitive for the detection of BYDV. Using one day instead uf the two-day procedure, the four viruses were still detectable and the ralative sensitivity of both techniques remained the same.

Whitefly Transmission and Efficient ssDNA Accumulation of Bean Golden Mosaic Geminivirus Require Functional Coat Protein

Bean golden mosaic geminivirus (BGMV) has a bipartite genome composed of two circular ssDNA components (DNA-A and DNA-B) and is transmitted by the whitefly, Bemisia tabaci. DNA-A encodes the viral replication proteins and the coat protein. To determine the role of BGMV coat protein systemic infection and whitefly transmission, two deletions and a restriction fragment inversion were introduced into the BGMV coat protein gene. All three coat protein mutants produced systemic infections when coinoculated with DNA-B onto Phaseolus vulgaris using electric discharge particle acceleration "particle gun." However, they were not sap transmissible and coat protein was not detected in mutant-infected plants. In addition, none of the mutants were transmitted by whiteflies. With all three mutants, ssDNA accumulation of DNA-A and DNA-B was reduced 25- to 50-fold and 3- to 10-fold, respectively, as compared to that of wild-type DNA. No effect on dsDNA-A accumulation was detected and there was 2- to 5-fold increase in dsDNA-B accumulation. Recombinants between the mutated DNA-A and DNA-B forms were identified when the inoculated coat protein mutant was linearized in the common region.

Depression among Chinese stroke survivors six months after discharge from a rehabilitation hospital

Aim and objective: The primary aim was to examine the prevalence of poststroke depression in Chinese stroke survivors six months after discharge from a rehabilitation hospital. A second aim was to determine whether six-month poststroke depression was associated with psychological, social and physical outcomes and demographic variables.---------- Background: There has been increasing recognition of the influence of depression on poststroke recovery. While some previous studies report associations between depression and social, psychological, physical and clinical outcomes, few studies had sufficient sample sizes for regression analysis thereby limiting the clinical applicability of their findings. ---------- Design: A cross-sectional design was used.---------- Method: Data were collected from 124 male and 86 female stroke survivors (mean age 71Æ7, SD 10Æ2 years). The Geriatric Depression Scale was used to measure depression, the State Self-esteem Scale to measure state self-esteem, the London Handicap Scale to measure participation restriction, the Social Support Questionnaire to measure satisfaction with social support and the Modified Barthel Index to measure functional ability. Results. Forty-two survivors (20Æ5%) reported mild and 33 (16Æ1%) reported severe depression. The presence of depression was associated with low levels of state self-esteem, social support satisfaction and functional ability. Logistic regression analysis revealed that these variables were statistically significant in predicting the probability of having depression (p < 0Æ05). ---------- Conclusions: Analyses in the present study revealed distinct patterns of correlates of depression, and the results were in agreement with prior studies that depression has a consistent positive ssociation with physical disability, living arrangements and social support and no significant association with the different types of brain lesion. Relevance to clinical practice. There is a need, routinely, to assess stroke survivors for depression and, where necessary, to intervene with the aim of enhancing psychological and social well-being.

Networks cities and ecological habitats

As the world’s rural populations continue to migrate from farmland to sprawling cities, transport networks form an impenetrable maze within which monocultures of urban form erupt from the spaces in‐between. These urban monocultures are as problematic to human activity in cities as cropping monocultures are to ecosystems in regional landscapes. In China, the speed of urbanisation is exacerbating the production of mono‐functional private and public spaces. Edges are tightly controlled. Barriers and management practices at these boundaries are discouraging the formation of new synergistic relationships, critical in the long‐term stability of ecosystems that host urban habitats. Some urban planners, engineers, urban designers, architects and landscape architects have recognised these shortcomings in contemporary Chinese cities. The ideology of sustainability, while critically debated, is bringing together thinking people in these and other professions under the umbrella of an ecological ethic. This essay aims to apply landscape ecology theory, a conceptual framework used by many professionals involved in land development processes, to a concept being developed by BAU International called Networks Cities: a city with its various land uses arranged in nets of continuity, adjacency, and superposition. It will consider six lesser‐known concepts in relation to creating enhanced human activity along (un)structured edges between proposed nets and suggest new frontiers that might be challenged in an eco‐city. Ecological theory suggests that sustaining biodiversity in regions and landscapes depends on habitat distribution patterns. Flora and fauna biologists have long studied edge habitats and have been confounded by the paradox that maximising the breadth of edges is detrimental to specialist species but favourable to generalist species. Generalist species of plants and animals tolerate frequent change in the landscape, frequenting two or more habitats for their survival. Specialist species are less tolerant of change, having specific habitat requirements during their life cycle. Protecting species richness then may be at odds with increasing mixed habitats or mixed‐use zones that are dynamic places where diverse activities occur. Forman (1995) in his book Land Mosaics however argues that these two objectives of land use management are entirely compatible. He postulates that an edge may be comprised of many small patches, corridors or convoluting boundaries of large patches. Many ecocentrists now consider humans to be just another species inhabiting the ecological environments of our cities. Hence habitat distribution theory may be useful in planning and designing better human habitats in a rapidly urbanising context like China. In less‐constructed environments, boundaries and edges provide important opportunities for the movement of multi‐habitat species into, along and from adjacent land use areas. For instance, invasive plants may escape into a national park from domestic gardens while wildlife may forage on garden plants in adjoining residential areas. It is at these interfaces that human interactions too flow backward and forward between land types. Spray applications of substances by farmers on cropland may disturb neighbouring homeowners while suburban residents may help themselves to farm produce on neighbouring orchards. Edge environments are some of the most dynamic and contested spaces in the landscape. Since most of us require access to at least two or three habitats diurnally, weekly, monthly or seasonally, their proximity to each other becomes critical in our attempts to improve the sustainability of our cities.

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families : functional analysis in heterozygous individuals

A protein-truncating variant of CHEK2, 1100delC, is associated with a moderate increase in breast cancer risk. We have determined the prevalence of this allele in index cases from 300 Australian multiple-case breast cancer families, 95% of which had been found to be negative for mutations in BRCA1 and BRCA2. Only two (0.6%) index cases heterozygous for the CHEK2 mutation were identified. All available relatives in these two families were genotyped, but there was no evidence of co-segregation between the CHEK2 variant and breast cancer. Lymphoblastoid cell lines established from a heterozygous carrier contained approximately 20% of the CHEK2 1100delC mRNA relative to wild-type CHEK2 transcript. However, no truncated CHK2 protein was detectable. Analyses of expression and phosphorylation of wild-type CHK2 suggest that the variant is likely to act by haploinsufficiency. Analysis of CDC25A degradation, a downstream target of CHK2, suggests that some compensation occurs to allow normal degradation of CDC25A. Such compensation of the 1100delC defect in CHEK2 might explain the rather low breast cancer risk associated with the CHEK2 variant, compared to that associated with truncating mutations in BRCA1 or BRCA2.

Multiple human single-stranded DNA binding proteins function in genome maintenance : structural, biochemical and functional analysis

DNA exists predominantly in a duplex form that is preserved via specific base pairing. This base pairing affords a considerable degree of protection against chemical or physical damage and preserves coding potential. However, there are many situations, e.g. during DNA damage and programmed cellular processes such as DNA replication and transcription, in which the DNA duplex is separated into two singlestranded DNA (ssDNA) strands. This ssDNA is vulnerable to attack by nucleases, binding by inappropriate proteins and chemical attack. It is very important to control the generation of ssDNA and protect it when it forms, and for this reason all cellular organisms and many viruses encode a ssDNA binding protein (SSB). All known SSBs use an oligosaccharide/oligonucleotide binding (OB)-fold domain for DNA binding. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating strand-exchange proteins and helicases, and mediation of protein–protein interactions. Recently two additional human SSBs have been identified that are more closely related to bacterial and archaeal SSBs. Prior to this it was believed that replication protein A, RPA, was the only human equivalent of bacterial SSB. RPA is thought to be required for most aspects of DNA metabolism including DNA replication, recombination and repair. This review will discuss in further detail the biological pathways in which human SSBs function.

Physical and functional interaction of the archaeal single-stranded DNA-binding protein SSB with RNA polymerase

Archaeal transcription utilizes a complex multisubunit RNA polymerase and the basal transcription factors TBP and TF(II)B, closely resembling its eukaryal counterpart. We have uncovered a tight physical and functional interaction between RNA polymerase and the single-stranded DNA-binding protein SSB in Sulfolobus solfataricus. SSB stimulates transcription from promoters in vitro under TBP-limiting conditions and supports transcription in the absence of TBP. SSB also rescues transcription from repression by reconstituted chromatin. We demonstrate the potential for promoter melting by SSB, suggesting a plausible basis for the stimulation of transcription. This stimulation requires both the single-stranded DNA-binding domain and the acidic C-terminal tail of the SSB. The tail forms a stable interaction with RNA polymerase. These data reveal an unexpected role for single-stranded DNA-binding proteins in transcription in archaea.

Hierarchical Bayesian models for estimating the extent of plant pest invasions

Plant biosecurity requires statistical tools to interpret field surveillance data in order to manage pest incursions that threaten crop production and trade. Ultimately, management decisions need to be based on the probability that an area is infested or free of a pest. Current informal approaches to delimiting pest extent rely upon expert ecological interpretation of presence / absence data over space and time. Hierarchical Bayesian models provide a cohesive statistical framework that can formally integrate the available information on both pest ecology and data. The overarching method involves constructing an observation model for the surveillance data, conditional on the hidden extent of the pest and uncertain detection sensitivity. The extent of the pest is then modelled as a dynamic invasion process that includes uncertainty in ecological parameters. Modelling approaches to assimilate this information are explored through case studies on spiralling whitefly, Aleurodicus dispersus and red banded mango caterpillar, Deanolis sublimbalis. Markov chain Monte Carlo simulation is used to estimate the probable extent of pests, given the observation and process model conditioned by surveillance data. Statistical methods, based on time-to-event models, are developed to apply hierarchical Bayesian models to early detection programs and to demonstrate area freedom from pests. The value of early detection surveillance programs is demonstrated through an application to interpret surveillance data for exotic plant pests with uncertain spread rates. The model suggests that typical early detection programs provide a moderate reduction in the probability of an area being infested but a dramatic reduction in the expected area of incursions at a given time. Estimates of spiralling whitefly extent are examined at local, district and state-wide scales. The local model estimates the rate of natural spread and the influence of host architecture, host suitability and inspector efficiency. These parameter estimates can support the development of robust surveillance programs. Hierarchical Bayesian models for the human-mediated spread of spiralling whitefly are developed for the colonisation of discrete cells connected by a modified gravity model. By estimating dispersal parameters, the model can be used to predict the extent of the pest over time. An extended model predicts the climate restricted distribution of the pest in Queensland. These novel human-mediated movement models are well suited to demonstrating area freedom at coarse spatio-temporal scales. At finer scales, and in the presence of ecological complexity, exploratory models are developed to investigate the capacity for surveillance information to estimate the extent of red banded mango caterpillar. It is apparent that excessive uncertainty about observation and ecological parameters can impose limits on inference at the scales required for effective management of response programs. The thesis contributes novel statistical approaches to estimating the extent of pests and develops applications to assist decision-making across a range of plant biosecurity surveillance activities. Hierarchical Bayesian modelling is demonstrated as both a useful analytical tool for estimating pest extent and a natural investigative paradigm for developing and focussing biosecurity programs.

Factors affecting somatic embryogenesis and transformation in modern plant breeding

Somatic embryogenesis and transformation systems are indispensable modern plant breeding components since they provide an alternative platform to develop control strategies against the plethora of pests and diseases affecting many agronomic crops. This review discusses some of the factors affecting somatic embryogenesis and transformation, highlights the advantages and limitations of these systems and explores these systems as breeding tools for the development of crops with improved agronomic traits. The regeneration of non-chimeric transgenic crops through somatic embryogenesis with introduced disease and pest-resistant genes for instance, would be of significant benefit to growers worldwide.

TaNF-YC11, one of the light-upregulated NF-YC members in Triticum aestivum, is co-regulated with photosynthesis-related genes

NF-Y is a heterotrimeric transcription factor complex. Each of the NF-Y subunits (NF-YA, NF-YB and NF-YC) in plants is encoded by multiple genes. Quantitative RT-PCR analysis revealed that five wheat NF-YC members (TaNF-YC5, 8, 9, 11 & 12) were upregulated by light in both the leaf and seedling shoot. Co-expression analysis of Affymetrix wheat genome array datasets revealed that transcript levels of a large number of genes were consistently correlated with those of the TaNF-YC11 and TaNF-YC8 genes in 3-4 separate Affymetrix array datasets. TaNF-YC11-correlated transcripts were significantly enriched with the Gene Ontology term photosynthesis. Sequence analysis in the promoters of TaNF-YC11-correlated genes revealed the presence of putative NF-Y complex binding sites (CCAAT motifs). Quantitative RT-PCR analysis of a subset of potential TaNF-YC11 target genes showed that ten out of the thirteen genes were also light-upregulated in both the leaf and seedling shoot and had significantly correlated expression profiles with TaNF-YC11. The potential target genes for TaNF-YC11 include subunit members from all four thylakoid membrane bound complexes required for the conversion of solar energy into chemical energy and rate limiting enzymes in the Calvin cycle. These data indicate that TaNF-YC11 is potentially involved in regulation of photosynthesis-related genes.