Unraveling the complexities of the Raf/MAP kinase pathway for pharmacological intervention

The Ras–MAP kinase pathway has attracted much attention from academic and pharmaceutical laboratories because of its central role in regulating tumor cell growth and survival, differentiation and angiogenesis. Although the central players in this pathway –Ras, Raf, and MEK – have been well studied, how best to exploit them for therapeutic gain has eluded oncology researchers in the past. Several small-molecule inhibitors that target specific steps of the MAP kinase cascade have recently entered the clinical arena. While we await answers on their ultimate therapeutic use, the availability of translational assays for monitoring target suppression will no doubt play a significant role in optimizing our chances of success.     

Unraveling the complexities of sphingosine-1-phosphate function: the mast cell model

Sphingosine-1-phosphate (S1P) is a lipid mediator involved in diverse biological processes, from vascular and neural development to the regulation of lymphocyte trafficking. Many of its functions are regulated by five widely expressed S1P G-protein-coupled receptors (S1P(1-5)). S1P is produced mostly intracellularly, thus, much of its potential as an autocrine and paracrine mediator depends on how, when, and where it is generated or secreted out of the cells. However, S1P can also have intracellular activity independent of its receptors, adding to the complexity of S1P function. The mast cell, a major effector cell during an allergic response, has proven instrumental towards understanding the complex regulation and function of S1P. Antigen (Ag) engagement of the IgE receptor in mast cells stimulates sphingosine kinases, which generate S1P and are involved in the activation of calcium fluxes critical for mast cell responses. In addition, mast cells secrete considerable amounts of S1P upon activation, thus affecting the surrounding tissues and recruiting inflammatory cells. Export of S1P is also involved in the autocrine transactivation of S1P receptors present in mast cells. The in vivo response of mast cells, however, is not strictly dependent on their ability to generate S1P, but they are also affected by changes in S1P in the environment previous to Ag challenge. This review will discuss the recent advances towards understanding the intricacies of S1P generation, secretion and regulation in mast cells. In addition, how S1P receptors are activated and their involvement in mast cell functions will also be covered, including new insights on the role of S1P in the mast cell-mediated allergic response of systemic anaphylaxis.     

Engineering the plastid genome of higher plants

The plastid genome of higher plants is an attractive target for engineering because it provides readily obtainable high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs and gene containment through the lack of pollen transmission. A chloroplast-based expression system that is suitable for the commercial production of recombinant proteins in tobacco leaves has been developed recently. This expression system includes vectors, expression cassettes and site-specific recombinases for the selective elimination of marker genes. Progress in expressing proteins that are biomedically relevant, in engineering metabolic pathways, and in manipulating photosynthesis and agronomic traits is discussed, as are the problems of implementing the technology in crops.     

PEG-mediated plastid transformation in higher plants

Summary Plastids are surrounded by an envelope consisting of a double membrane. This barrier has to be penetrated or overcome by the DNA when transforming the plastome. Both the biolistic and polyethylene glycol-mediated transformation techniques accomplish this task, albeit by different mechanisms. We were the first laboratory to successfully use the polyethylene glycol (PEG)-method for plastid transformation, yet we use the particle gun when appropriate. In this report we compare the two methods and discuss their shortcomings and advantages. Plastid transformations with various constructs, mainly using theaadA gene as a selective marker, were performed. We point to potential problems likely to be encountered during the transformation and selection processes and offer possibilities for improvement. We give further examples of the successful application of plastome transformation and show its merits in addressing biological questions concerning the elucidation of plastid sequences of unknown function and the control of plastid gene expression. Based on a presentation in the symposium “Organelle Transformation” during the 1997 SIVB Congress held in Washington, DC June 14–18, 1997. An erratum to this article is available at .     

Unraveling salt stress signaling in plants

Salt stress is a major environmental factor limiting plant growth and productivity. A better understanding of the mechanisms mediating salt resistance will help researchers design ways to improve crop performance under adverse environmental conditions. Salt stress can lead to ionic stress, osmotic stress and secondary stresses, particularly oxidative stress, in plants. Therefore,to adapt to salt stress, plants rely on signals and pathways that re-establish cellular ionic, osmotic, and reactive oxygen species(ROS) homeostasis. Over the past two decades, genetic and biochemical analyses have revealed several core stress signaling pathways that participate in salt resistance. The Salt Overly Sensitive signaling pathway plays a key role in maintaining ionic homeostasis,via extruding sodium ions into the apoplast. Mitogenactivated protein kinase cascades mediate ionic, osmotic,and ROS homeostasis. SnR K2(sucrose nonfermenting1-related protein kinase 2) proteins are involved in maintaining osmotic homeostasis. In this review, we discuss recent progress in identifying the components and pathways involved in the plant's response to salt stress and their regulatory mechanisms. We also review progress in identifying sensors involved in salt-induced stress signaling in plants.     

Unraveling the functions of glycosyltransferase family 47 in plants

The function of glycosyltransferases (GTs) from family GT47 was first identified in animal exostosins as β-glucuronyltransferase involved in the synthesis of heparan sulfate. Two recent papers report the functions of two plant members in this family as a pectin β-glucuronyltransferase and a xyloglucan β-galactosyltransferase. These findings greatly extend our understanding of the biological functions of family GT47 and also represent an important leap toward the molecular dissection of cell wall biosynthesis.     

Unraveling the role of mitochondria during oxidative stress in plants

The sedentary habit of plants means that they must stand and fight environmental stresses that their mobile animal cousins can avoid. A range of these abiotic stresses initiate the production in plant cells of reactive oxygen and nitrogen species that ultimately lead to oxidative damage affecting the yield and quality of plant products. A complex network of enzyme systems, producing and quenching these reactive species operate in different organelles. It is the integration of these compartmented defense systems that coordinates an effective response to the various stresses. Future attempts to improve plant growth or yield must consider the complexity of inter-organelle signaling and protein targeting if they are to be successful in producing plants with resistance to a broad range of stresses. Here we highlight the role of pre-oxidant, antioxidant, and post-oxidant defense systems in plant mitochondria and the potential role of proteins targeted to both mitochondria and chloroplasts, in an integrated defense against oxidative damage in plants.     

The molecular biology of plastid division in higher plants

Plastids are essential plant organelles vital for life on earth, responsible not only for photosynthesis but for many fundamental intermediary metabolic reactions. Plastids are not formed de novo but arise by binary fission from pre-existing plastids, and plastid division therefore represents an important process for the maintenance of appropriate plastid populations in plant cells. Plastid division comprises an elaborate pathway of co-ordinated events which include division machinery assembly at the division site, the constriction of envelope membranes, membrane fusion and, ultimately, the separation of the two new organelles. Because of their prokaryotic origin bacterial cell division has been successfully used as a paradigm for plastid division. This has resulted in the identification of the key plastid division components FtsZ, MinD, and MinE, as well as novel proteins with similarities to prokaryotic cell division proteins. Through a combination of approaches involving molecular genetics, cell biology, and biochemistry, it is now becoming clear that these proteins act in concert during plastid division, exhibiting both similarities and differences compared with their bacterial counterparts. Recent efforts in the cloning of the disrupted loci in several of the accumulation and replication of chloroplasts mutants has further revealed that the division of plastids is controlled by a combination of prokaryote-derived and host eukaryote-derived proteins residing not only in the plastid stroma but also in the cytoplasm. Based on the available data to date, a working model is presented showing the protein components involved in plastid division, their subcellular localization, and their protein interaction properties.     

The genetic control of plastid division in higher plants

The division of plastids is an important part of plastid differentiation and development and in distinct cell types, such as leaf mesophyll cells, results in large populations of chloroplasts. The morphology and population dynamics of plastid division have been well documented, but the molecular controls underlying plastid division are largely unknown. With the isolation of Arabidopsis mutants in which specific aspects of plastid and proplastid division have been disrupted, the potential exists for a detailed knowledge of how plastids divide and what factors control the rate of division in different cell types. It is likely that knowledge of plant homologues of bacterial cell division genes will be essential for understanding this process in full. The processes of plastid division and expansion appear to be mutually independent processes, which are compensatory when either division or expansion are disrupted genetically. The rate of cell expansion appears to be an important factor in initiating plastid division and several systems involving rapid cell expansion show high levels of plastid division activity. In addition, observation of plastids in different cell types in higher plants shows that cell-specific signals are also important in the overall process in determining not only the differentiation pathway of plastids but also the extent of plastid division. It appears likely that with the exploitation of molecular techniques and mutants, a detailed understanding of the molecular basis of plastid division may soon be a reality.