Functional EF-hands in neuronal calcium sensor GCAP2 determine its phosphorylation state and subcellular distribution in vivo, and are essential for photoreceptor cell integrity

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca2+-free and Ca2+-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca2+ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca2+. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF(-)GCAP2), we show that GCAP2 locked in its Ca2+-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca2+-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF(-)GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca2+-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca2+ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in 'equivalent-light'' scenarios.

Identification of Albizia lebbeck seed coat chitin-binding vicilins (7S globulins) with high toxicity to the larvae of the bruchid Callosobruchus maculatus

Seed coat is a specialized maternal tissue that interfaces the embryo and the external environment during embryogenesis, dormancy and germination. In addition, it is the first defensive barrier against penetration by pathogens and herbivores. Here we show that Albizia lebbeck seed coat dramatically compromises the oviposition, eclosion and development of the bruchid Callosobruchus maculatus. Dietary supplementation of bruchid larvae with A. lebbeck seed coat flour causes severe weight loss and reduces survival. By means of protein purification, mass spectrometry and bioinformatic analyses, we show that chitin-binding vicilins are the main source of A. lebbeck tegumental toxicity to C. maculatus. At concentrations as low as 0.1%, A. lebbeck vicilins reduce larval mass from 8.1 ± 1.7 (mass of control larvae) to 1.8 ± 0.5 mg, which corresponds to a decrease of 78%. Seed coat toxicity constitutes an efficient defense mechanism, hindering insect predation and preventing embryo damage. We hypothesize that A. lebbeck vicilins are good candidates for the genetic transformation of crop legumes to enhance resistance to bruchid predation.

The Elucidation of the involvement of endonuclease DNase y in reducing DNA transfection efficiency in mammalian cells

Gene therapy is predicated upon efficient gene transfer. While viral vectors are the method of choice for transformation efficiency, the immunogenicity and safety concerns remain problematic. Non-viral vectors, on the other hand, have shown high degrees of safety and are mostly non-immunogenic in nature. However, non-viral vectors usually suffer from low levels oftransformation efficiency and transgene expression. Thus, increasing transformation efficiency ofnon-viral vectors, in particular by calcium phosphate co-precipitation technique, is a way of generating a suitable vector for gene therapy and is the aim of this study. It is a long known fact that different cell lines have different transfection efficiencies regardless oftransfection methodology (Lin et a!., 1994). Using commonly available cell lines Madine-Darby Bovine Kidney (MDBK), HeLa and Human Embryonic Kidney (HEK-293), we have shown a decreasing trend ofDNase activity based on a plasmid digestion assay. From densitometry studies, as much as a 40% reduction in DNase activity was observed when comparing HEK-293 (least active) to MDBK (most active). Using various biochemical assays, it was determined that DNase y, in particular, was expressed more highly in MDBK cells than both HeLa and HEK-293. Upon cloning of the bovine DNase y gene, we utilized the sequence information to construct antisense expressing plasmids via both traditional antisense RNA (pASDGneoM) and siRNA (psiRNA-S4, psiRNA-S11 and psiRNA-S16). For the construction ofpASDGneoM, the 3' end of the DNase y was inserted in opposite orientation under a cytomegalovirus (CMV) promoter such that the expression ofRNA complementary to the DNase 2 ymRNA occurred. For siRNA plasmids, the sequence was screened to yield optimal short sequences for siRNA inhibition. The silencing ofbovine DNase y led to an increase in transfection efficiency based on traditional calcium phosphate co-precipitation technique; stable clones of siRNA-producing MDBK cell lines (psiRNA-S4 Bland psiRNA-S4 B4) both demol).strated 4-fold increases in transfection efficiency. Furthermore, serial transfection of antisense DNase y plasmid pASDGneoM and reporter pCMV-~ showed a maximum of 8-fold increase in transfection efficiency when the two separate transfections were carried out 4 hours apart (i.e. transfection ofpASDGneoM, separated by four hours, then transfection ofpCMV-~). Together, these results demonstrate the involvement ofDNase y in reducing transfection efficiency, at least by traditional calcium phosphate technique.

Transfection of newt blastema mesenchyme using the techniques of lipofection and direct gene transfer

The regenerating amphibian limb provides a useful system for studying genes involved in the establishment of positional information. While a number of candidate genes that may playa role in pattern formation have been identified, their function in vivo is unknown in this system. To better ascertain the role of these genes, it would be useful to be able to alter their normal patterns of expression in vivo and to assess the effects of this misexpression on limb pattern. In order to achieve this, a method of introducing a plasmid containing the eDNA of a gene of interest into a newt blastema (a growth zone of mesenchymal progenitor cells) is needed. Unfortunately, most commonly used transfection techniques cannot be used with newt blastema cells. In this study, I have used the techniques of lipofection and direct gene transfer to introduce plasmid DNA containing reporter genes into the cells of a regenerating newt limb. The technique of lipofection was most effective when the blastema cells were transfected in vitro. The optimal ratio for transfection was shown to be 1:3 DNA:Lipofectin (W/w) , and an increase in the amount of DNA present in the mixture (1:3 ratio maintained) resulted in a corresponding increase in gene expression. The technique of direct gene transfer was used to transfect newt blastema cells with and without prior complex formation with Lipofectin. Injection of plasmid DNA alone provided the most 3 promising results. It was possible to introduce plasmid DNA containing the reporter gene ~-galactosidase and achieve significant gene expression in cells associated with the injection site. In the future, it would be interesting to use this technique to inject plasmid DNA containing a gene which may have a role in pattern formation into specific areas of the newt blastema and to analyze the resulting limb pattern that emerges.

Droits de propriété intellectuelle et protection des plantes: approches, limites et perspectives

Le droit de la propriété intellectuelle présente, depuis quelques années, un intérêt particulier à l'évolution de la recherche sur les plantes. Ceci s'est traduit, au plan international, par l'adoption de plusieurs instruments visant à assurer une meilleure protection des investissements consentis dans ce domaine. Il s'agit notamment de la Convention de l'UPOV, qui s'inscrit dans une logique de protection par la voie sui generis avec la possibilité de délivrance de certificat d'obtention végétale aux sélectionneurs; de l'Accord ADPIC, qui, en plus de recommander un système sui generis efficace, ouvre l'option de protection par brevet ou en définitive par le cumul des deux systèmes; de la Convention sur la Diversité Biologique (CDB) et du Traité de la FAO portant sur les ressources phytogénétiques pour l'alimentation et l'agriculture, qui, favorables aux deux précédentes formes de protection, demandent que soient prises en compte des considérations relatives aux droits souverains des pays sur leurs ressources végétales, au partage des bénéfices, etc. Au plan régional, on distingue, entre autres, l'initiative de l'Afrique, visant à assurer la protection des plantes suivant une logique partagée entre l'alignement sur les normes internationales existantes (Accord de Bangui) ou l'institution d'une autre législation originale qui reflète les réalités et préoccupations du continent (Loi modèle). Il apparaît donc qu'il existe plusieurs instruments pour cerner la même réalité. Ceci est forcément la source de quelques difficultés qui sont d'ordre conceptuel, socioéconomique, environnemental et juridique. Pour les pallier, il est important que certaines conditions soient satisfaites afin d'harmoniser les points de vue entre les différents acteurs concernés par la question et d'assurer une appropriation conséquente des instruments adoptés.

A survey of wheat landraces in Oman

Little is known about the diversity of wheat (Triticum spp.) in Oman. Therefore, a survey was conducted in northern Oman to collect landraces of Triticum durum, T. aestivum and T. dicoccon for subsequent morphological characterization and investigations on stress adaptation. The results show that the cultivation of these landraces (the genetic composition of which remains to be studied in more detail) is done primarily by traditional farmers who preserve the inherited germplasm on often tiny plots in remote mountain oases. This type of traditional cultivation is under heavy economic pressure. An appendix of landraces of other crops collected in the Batinah region and in the mountain oases can be found online.

Los Intereses geoestatégicos de Rusia, Brasil y Estados Unidos en la Cuenca Amazónica periodo 2004-2008

La Cuenca Amazónica es el nuevo escenario geoestratégico redefinido de Halford Mackinder: el Green Heart Land, el cual refleja los intereses geoestratégicos de diferentes potencias mundiales como Brasil, Rusia y Estados Unidos, los cuales actuan bajos sus propios intereses nacionales. Su interacción deja como consecuencia la elaboración de una nueva estructura en el Sistema Internacional bajo nuevas nociones de seguridad, las cuales dinamizan las fichas del ajedrez mundial descrito por Zbniew Brzezinski.

Patents and transgenic plants

One of the recurring themes in any discussion concerning the application of genetic transformation technology is the role of Intellectual Property Rights (IPR). This term covers both the content of patents and the confidential expertise, usually related to methodology and referred to as “Trade Secrets”. This review will explain the concepts behind patent protection, and will discuss the wide-ranging scope of existing patents that cover all aspects of transgenic technology, from selectable markers and novel promoters to methods of gene introduction. Although few of these patents have any significant commercial value, there are a small number of key patents that may restrict the “freedom to operate” of any company seeking to exploit the methods. Over the last twenty years, these restrictions have forced extensive cross-licensing between ag-biotech companies and have been one of the driving forces behind the consolidation of these companies. Although such issues are often considered to be of little interest to the academic scientist working in the public sector, they are of great importance in any debate about the role of “public-good breeding” and of the relationship between the public and private sectors.

Multiple domestications and their taxonomic consequences: the example of Phaseolus vulgaris

The evolutionary history of P. vulgaris is important to those working on its genetic resources, but is not reflected in its infraspecific taxonomy. Genetic isolation of wild populations between and also within Middle and South America has resulted in morphological and molecular differentiation. Populations from northern and southern ends of the range are assigned to different gene pools, though intermediates occur in intervening areas. Chloroplast haplotypes suggest three distinct lineages of wild beans and several intercontinental dispersals. The species was domesticated independently in both Middle and South America, probably several times in Middle America. This, together with further differentiation under human selection, has produced distinct races among domesticated beans. The informal categories of wild versus domesticated, gene pool, and race convey the evolutionary picture more clearly than the formal categories provided by the Codes of Nomenclature for wild or cultivated plants.

Sainfoin (Onobrychis viciifolia): a beneficial forage legume

The Onobrychis genus comprises a few agronomically important forage legume species, with sainfoin (Onobrychis viciifolia) being the most widespread. O. viciifolia has a long history of traditional culture worldwide, but its use has declined in western countries over the last decades. It suffers from low productivity and is more difficult to maintain than other legumes but is known to have valuable characteristics such as palatability and drought tolerance. Recent studies suggest that it has several other highly beneficial properties due to its unique tannin and polyphenol composition. Condensed tannins present in Onobrychis species have been shown to confer anthelmintic properties, increase protein utilization and prevent bloating; they may also have the potential to reduce greenhouse gas emissions. Positive effects on wildlife and honey production could also be advantageous in the context of sustainable farming. Modern breeding programmes have not been a priority, leading to a lack of genetic knowledge in comparison to extensively used forage legumes. It is expected that potential for O. viciifolia improvements could be achieved by rigorous characterization of the available germplasm and utilization of characters derived from close relatives of the genus. Breeding priorities for the future would include enhanced germination and improved early establishment, allied to the best anthelmintic properties observed in some varieties.

Farmers' management of rice varietal diversity in the mid-hills of Nepal: implications for on-farm conservation and crop improvement

Season-long monitoring of on-farm rice (Oryza sativa, L.) plots in Nepal explored farmers' decision-making process on the deployment of varieties to agroecosystems, application of production inputs to varieties, agronomic practices and relationship between economic return and area planted per variety. Farmers deploy varieties [landraces (LRs) and modern varieties (MVs)] to agroecosystems based on their understanding of characteristics of varieties and agroecosystems, and the interaction between them. In marginal growing conditions, LRs can compete with MVs. Within an agroecosystem, economic return and area planted to varieties have positive relationship, but this is not so between agroecosystems. LRs are very diverse on agronomic and economic traits; therefore, they cannot be rejected a priori as inferior materials without proper evaluation. LRs have to be evaluated for useful traits and utilized in breeding programmes to generate farmer-preferred materials for marginal environments and for their conservation on-farm.

Patents for plants: context and current status

One of the important themes in any discussion concerning the application of genetic transformation technology in horticulture or elsewhere is the role of Intellectual Property Rights (IPR). This term covers both the content of patents and the confidential expertise, usually related to methodology and referred to as “Trade Secrets”. This review will explain the concepts behind patent protection, and will discuss the wide-ranging scope of existing patents that cover novel genotypes of plants as well as all aspects of transgenic technology, from selectable markers and novel promoters to methods of gene introduction. Although few of these patents have any significant commercial value there are a small number of key patents that may restrict the “freedom to operate” of any company seeking to exploit the methods in the production of transgenic varieties. Over the last twenty years, these restrictions have forced extensive cross-licensing between ag-biotech companies and have been one of the driving forces behind the consolidation of these companies. Although such issues may have limited relevance in the horticultural sector, and are often considered to be of little interest to the academic scientist working in the public sector, they are of great importance in any debate about the role of “public-good breeding” and of the relationship between the public and private sectors.

Institutional capacity and implementation issues in farmers' rights

Armed with the ‘equity’ and ‘conservation’ arguments that have a deep resonance with farming communities, developing countries are crafting a range of measures designed to protect farmers’ access to innovations, reward their contributions to the conservation and enhancement of plant genetic resources and provide incentives for sustained on-farm conservation. These measures range from the commericialization of farmers’ varieties to the conferment of a set of legally enforceable rights on farming communities – the exercise of which is expected to provide economic rewards to those responsible for on-farm conservation and innovation. The rights-based approach has been the cornerstone of legislative provision for implementing farmers’ rights in most developing countries. In drawing up these measures, developing countries do not appear to have systematically examined or provided for the substantial institutional capacity required for the effective implementation of farmers’ rights provisions. The lack of institutional capacity threatens to undermine any prospect of serious implementation of these provisions. More importantly, the expectation that significant incentives for on-farm conservation and innovation will flow from these ‘rights’ may be based on a flawed understanding of the economics of intellectual property rights. While farmers’ rights may provide only limited rewards for conservation, they may still have the effect of diluting the incentives for innovative institutional breeding programs – with the private sector increasingly relying on non-IPR instruments to profit from innovation. The focus on a rights-based approach may also draw attention away from alternative stewardship-based approaches to the realization of farmers’ rights objectives.