Whole-embryo culture of Drosophila : development of embryonic tissues in vitro

Summary We have devised techniques to culture whole, dissected embryos of Drosophila melanogaster. We examine multiple aspects of the morphological and physiological development of the epidermis, musculature, nervous system, and internal organs in this cultured preparation, and show that in vitro development closely parallels normal embryogenesis. These techniques permit a wide range of experimental manipulations during embryogenesis and allow us to extend observations through late embryonic stages, after cuticle deposition. Applications of this technique are presented.     

Abscisic acid biosynthesis during corn embryo development

In this study we examined the biosynthesis of abscisic acid (ABA) by developing corn (Zea mays L.) embryos. Three comparisons were made: ABA biosynthesis in embryos isolated from kernels grown in vitro with those grown in the field; the developmental profile of ABA content with that of biosynthesis; and ABA biosynthesis in corn embryos lacking carotenoid precursors with ABA biosynthesis in normal embryos. Embryos were harvested at various times during seed development and divided into two groups. Endogenous levels of ABA were measured in one group of embryos and ABA biosynthetic capacity was measured in the other group. The ABA biosynthetic capacity was measured with and without tetcyclacis (an inhibitor of ABA degradation) in embryos from both field-grown and in-vitro-grown corn kernels. Reduced-carotenoid (either fluridone-treated or genetically viviparous) embryos were also included in the study. Corn kernels developing under field and in-vitro conditions differed from each other in their responses to tetcyclacis and in their profiles of ABA biosynthesis during development. Therefore, in-vitro kernel culture may not be an appropriate substitute for field conditions for studies of embryo development. The developmental profiles of endogenous ABA content differed from those of ABA biosynthesis in isolated embryos of both in-vitro-and field-grown kernels. This indicated that ABA levels in the developing embryos were determined by import from the maternal tissues available to the embryos rather than by in-situ biosynthesis. In embryos with reduced levels of carotenoids, either fluridone-treated or genetically viviparous embryos, ABA biosynthesis was low or nonexistent. This result is expected for the presence of an indirect pathway of ABA biosynthesis and in the absence of ABA precursors.Abbreviations ABA abscisic acid - DAP days after pollination     

Betulinic and oleanolic acids isolated from Forsythia suspensa Vahl inhibit urease activity of Helicobacter pylori

Sixteen medicinal herbs were selected from a database on traditional herbal materials as well as literature on Korean plant resources. Then ethanol (70%, v/v) extracts of these herbs were tested for inhibition of the urease activity of Helicobacter pylori. The urease activity of H. pylori was strongly (82%) inhibited by extract of Forsythia suspensa Vahl. Active compounds in extract of Forsythia suspensa Vahl were first separated by batch mode solvent extraction, followed by purification by silica gel and octadecyl silica gel column chromatography using solvents of different polarity. According to NMR analysis of the last chromatographic fraction, we identified the presence of betulinic acid and oleanolic acid, which are known to have anti-inflammatory, anti-cancer, and anti-HIV viral activities.     

Morphometric analysis of the genusHemerocallis L. (Liliaceae) in Korea

To better understand the patterns of variability and distributions ofHemerocallis in Korea, 53 locations were visited and measurements of 19 morphological and phenological characters were taken on plants directly from their natural habitats. For morphometric analysis, 10 plants from each of 34 populations and five herbarium specimens ofH. middendorffii were used and the data from 12 quantitative characters was analyzed using univariate analysis. Except the littoral populations of Cheju, Hong, Taehuksan, and Sohuksan Islands (H. hongdoensis M. Chung & S. Kang), three peninsular KoreanHemerocallis species can be recognized mainly in South Korea:H. hakuunensis Nakai (=H. micrantha Nakai, growing on southern, central, and northwestern Korea);H. thunbergii Baker (=H. coreana Nakai, found on southeastern and central Korea); andH. middendorffii Tr. et Mey. (central and northeastern Korea). Morphological and phenological features contributing to recognition of the three groups were; color of perianth, shape of roots, shape of inflorescence, flowering time, odor, length of inflorescence, width of the lowest bracts, length of perianth tube enclosing a ovary, width of the inner perianth lobes. Natural hybridization seems to be rare in KoreanHemerocallis. It appears that the KoreanHemerocallis species are relatively well characterized by their distribution patterns, phenology, and habitats compared with the JapaneseHemerocallis species.     

Optimized production of lignolytic manganese peroxidase in immobilized cultures of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Manganese peroxidase (MnP) is a key enzyme involved in the lignolysis of white-rot fungi. The purpose of this study is to investigate the effect of immobilization and culture conditions on MnP production in cultures of Phanerochaete chrysosporium grown on polyurethane foam. Higher concentrations of foam and lower levels of spore inoculums resulted in the formation of scattered mycelial pellets, increased autolysis of chlamydospore-like cells (a reservoir of MnP), and a higher activity of MnP. Even though MnP was a secondary metabolite, the addition of 5 times more glucose and diammonium tartrate, as carbon and nitrogen sources, resulted in a 4 fold increase in the dry cell mass. However, MnP activity decreased under these conditions to less than half, due to the formation of increasingly dense pellets and the inhibited lysis of chlamydospore-like cells.     

Nitrogen assimilation in field-grown winter wheat: Direct measurements of nitrate reduction in roots using15N

Summary Nitrate fertiliser labelled with¹⁵N was applied to a field grown crop of winter wheat. Uptake and assimilation of fertiliser nitrate was studied by monitoring the appearance of labelled nitrate and labelled amino acids in the xylem sap. Shortly after applying¹⁵N-nitrate to the soil about 30 per cent of recently absorbed¹⁵N was in the reduced form, indicating that roots of cereal crops can make a substantial contribution in reducing nitrate. Seasonal changes in crop growth andin vivo NRA are also described.     

Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.     

A set of epitope-tagging integration vectors for functional analysis in Saccharomyces cerevisiae

Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.