Genetic analysis of chromosomal rearrangements in the cyclops region of the zebrafish genome.

Genetic screens in zebrafish have provided mutations in hundreds of genes with essential functions in the developing embryo. To investigate the possible uses of chromosomal rearrangements in the analysis of these mutations, we genetically characterized three gamma-ray induced alleles of cyclops (cyc), a gene required for development of midline structures. We show that cyc maps near one end of Linkage Group 12 (LG 12) and that this region is involved in a reciprocal translocation with LG 2 in one gamma-ray induced mutation, cyc(b213). The translocated segments together cover approximately 5% of the genetic map, and we show that this rearrangement is useful for mapping cloned genes that reside in the affected chromosomal regions. The other two alleles, cyc(b16) and cyc(b229), have deletions in the distal region of LG 12. Interestingly, both of these mutations suppress recombination between genetic markers in LG 12, including markers at a distance from the deletion. This observation raises the possibility that these deletions affect a site required for meiotic recombination on the LG 12 chromosome. The cyc(b16) and cyc(b229) mutations may be useful for balancing other lethal mutations located in the distal region of LG 12. These results show that chromosomal rearrangements can provide useful resources for mapping and genetic analyses in zebrafish.     

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

The Discovery of orally available thrombin inhibitors : Studies towards the optimisation of CGH1668

The chemical optimisation of CGH1668 1 is described employing an in vivo model of absorption to determine the influence on bioavailability of single point modifications to five key molecular templates. The discovery of an orally bioavailable and selective thrombin inhibitor, 24, highlights the utility of this approach.     

Cell Cycle–Mediated Regulation of Plant Infection by the Rice Blast Fungus

To gain entry to plants, many pathogenic fungi develop specialized infection structures called appressoria. Here, we demonstrate that appressorium morphogenesis in the rice blast fungus Magnaporthe oryzae is tightly regulated by the cell cycle. Shortly after a fungus spore lands on the rice (Oryza sativa) leaf surface, a single round of mitosis always occurs in the germ tube. We found that initiation of infection structure development is regulated by a DNA replication-dependent checkpoint. Genetic intervention in DNA synthesis, by conditional mutation of the Never-in-Mitosis 1 gene, prevented germ tubes from developing nascent infection structures. Cellular differentiation of appressoria, however, required entry into mitosis because nimA temperature-sensitive mutants, blocked at mitotic entry, were unable to develop functional appressoria. Arresting the cell cycle after mitotic entry, by conditional inactivation of the Blocked-in-Mitosis 1 gene or expression of stabilized cyclinB-encoding alleles, did not impair appressorium differentiation, but instead prevented these cells from invading plant tissue. When considered together, these data suggest that appressorium-mediated plant infection is coordinated by three distinct cell cycle checkpoints that are necessary for establishment of plant disease.     

An Approach to Integrating Environmental Considerations Within Managerial Decision‐Making

Recent environmental trends, including (1) an expansion of existing command and control directives, (2) the introduction of market‐based policy instruments, and (3) the adoption of extended producer responsibility, have created a need for new tools to help managerial decision‐making. To address this need, we develop a nonlinear mathematical programming model from a profit‐maximizing firm's perspective, which can be tailored as a decision‐support tool for firms facing environmental goals and constraints. We typify our approach using the specific context of diesel engine manufacturing and remanufacturing. Our model constructs are based on detailed interviews with top managers from two leading competitors in the medium and heavy‐duty diesel engine industry. The approach allows the incorporation of traditional operations‐planning considerations—in particular, capacity, production, and inventory—together with environmental considerations that range from product design through production to product end of life. A current hurdle to implementing such a model is the availability of input data. We therefore highlight the need not only to involve all departments within businesses but also for industrial ecologists and business managers to work together to implement meaningful decision models that are based on accurate and timely data and can have positive economic and environmental impact.     

Serotonin catabolism in the central and enteric nervous systems of rats upon induction of serotonin syndrome

Serotonin, a well-known neurotransmitter in mammals, has been linked to a number of neurological and gastrointestinal disorders. One of these disorders, serotonin syndrome, is a potentially deadly condition caused by increased levels of serotonin in the extracellular space. Information on the neurochemical effects of serotonin syndrome on serotonin catabolism is lacking, particularly in relation to the enteric system of the gastrointestinal tract. Here the catabolism of serotonin is monitored in rats with pharmacologically induced serotonin syndrome, with the catabolites characterized using a specialized capillary electrophoresis system with laser-induced native fluorescence detection. Animals induced with serotonin syndrome demonstrate striking increases in the levels of serotonin and its metabolites. In the brain, levels of serotonin increased 2- to 3-fold in animals induced with serotonin syndrome. A major serotonin metabolite, 5-hydroxyindole acetic acid, increased 10- to 100-fold in experimental animals. Similar results were observed in the gastrointestinal tissues; in the small intestines, serotonin levels increased 4- to 5-fold. Concentrations of 5-hydroxyindole acetic acid increased 32- to 100-fold in the intestinal tissues of experimental animals. Serotonin sulfate showed surprisingly large increases, marking what may be the first time the compound has been reported in rat intestinal tissues.     

Tps1 regulates the pentose phosphate pathway, nitrogen metabolism and fungal virulence

Trehalose fulfils a wide variety of functions in cells, acting as a stress protectant, storage carbohydrate and compatible solute. Recent evidence, however, indicates that trehalose metabolism may exert important regulatory roles in the development of multicellular eukaryotes. Here, we show that in the plant pathogenic fungus Magnaporthe grisea trehalose-6-phosphate (T6P) synthase (Tps1) is responsible for regulating the pentose phosphate pathway, intracellular levels of NADPH and fungal virulence. Tps1 integrates glucose-6-phosphate (G6P) metabolism with nitrogen source utilisation, and thereby regulates the activity of nitrate reductase. Activity of Tps1 requires an associated regulator protein Tps3, which is also necessary for pathogenicity. Tps1 controls expression of the nitrogen metabolite repressor gene, NMR1, and is required for expression of virulence-associated genes. Functional analysis of Tps1 indicates that its regulatory functions are associated with binding of G6P, but independent of Tps1 catalytic activity. Taken together, these results demonstrate that Tps1 is a central regulator for integration of carbon and nitrogen metabolism, and plays a pivotal role in the establishment of plant disease.     

alphaII-spectrin is essential for assembly of the nodes of Ranvier in myelinated axons

Saltatory conduction in myelinated axons requires organization of the nodes of Ranvier, where voltage-gated sodium channels are prominently localized [1]. Previous results indicate that alphaII-spectrin, a component of the cortical cytoskeleton [2], is enriched at the paranodes [3, 4], which flank the node of Ranvier, but alphaII-spectrin's function has not been investigated. Starting with a genetic screen in zebrafish, we discovered in alphaII-spectrin (alphaII-spn) a mutation that disrupts nodal sodium-channel clusters in myelinated axons of the PNS and CNS. In alphaII-spn mutants, the nodal sodium-channel clusters are reduced in number and disrupted at early stages. Analysis of chimeric animals indicated that alphaII-spn functions autonomously in neurons. Ultrastructural studies show that myelin forms in the posterior lateral line nerve and in the ventral spinal cord in alphaII-spn mutants and that the node is abnormally long; these findings indicate that alphaII-spn is required for the assembly of a mature node of the correct length. We find that alphaII-spectrin is enriched in nodes and paranodes at early stages and that the nodal expression diminishes as nodes mature. Our results provide functional evidence that alphaII-spectrin in the axonal cytoskeleton is essential for stabilizing nascent sodium-channel clusters and assembling the mature node of Ranvier.