Efficient expression and primary purification of 6-his tagged human Fas ligand in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Human Fas ligand (hFasL) is a member of the tumor necrosis factor (TNF) family with many medical interests. To produce this protein efficiently, an improved vector which could express the recombinant hFasL protein with a 6-his tag at its C-terminal was constructed. The new vector was transformed into Dictyostelium discoideum AX3 which then produced 157 μg hFasL l⁻¹. Using one-step Ni-affinity chromatography, it was purified with a recovery of 92% and purity of 91%.     

Analysis of the behaviour of<Emphasis Type="Italic"> Dictyostelium discoideum</Emphasis> in immobilised state by means of continuous cultivation

The social amoeba Dictyostelium discoideum is amenable to cultivation in the immobilised state most simply by colonisation of porous supports. An analysis of the growth behaviour of D. discoideum in the immobilised state is reported. For this purpose, D. discoideum was cultivated in continuously operated reactors in a suspension culture (homogeneous system) and immobilised on a porous support (heterogeneous system). Thus, it is possible to compare homogeneous and heterogeneous systems under steady-state conditions. Immobilisation was achieved by the colonisation of porous glass beads (SIRAN). Simple models are applied in order to describe the growth behaviour of fractions of both the cells in free suspension and the cells inside the porous carrier. This analysis shows that D. discoideum inside the pores grows at a rate of only about 10% compared with that in free solution. The consequence of this behaviour is discussed in terms of reactor performance.     

Cultivation of immobilized <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> for the production of soluble human Fas ligand

Dictyostelium discoideum was immobilized by cultivation on inorganic porous matrices consisting of broken pumice or a ceramic catalyst carrier (CeramTec) to produce human soluble Fas ligand (hFasL). These supports were actively colonized by D. discoideum reaching cell (number) densities 10–20 times higher locally than those observed in suspension culture under similar conditions. In repeated batch or continuous operation, hFasL productivities of up to 15–25 g h^–1 l^–1 pore volume were attained. The immobilized cell densities and hFasL productivities could be kept constant for a long period of time by repeated renewal or continuous feeding of complex or synthetic medium.     

Laboratory-scale biofiltration of acrylonitrile by <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> DAP 96622 in a trickling bed bioreactor

Acrylonitrile (ACN), a volatile component of the waste generated during the production of acrylamide, also is often associated with aromatic contaminants such as toluene and styrene. Biofiltration, considered an effective technique for the treatment of volatile hydrocarbons, has not been used to treat volatile nitriles. An experimental laboratory-scale trickling bed bioreactor using cells of Rhodococcus rhodochrous DAP 96622 supported on granular activated carbon (GAC) was developed and evaluated to assess the ability of biofiltration to treat ACN. In addition to following the course of treatability of ACN, kinetics of ACN biodegradation during both recycle batch and open modes of operation by immobilized and free cells were evaluated. For fed-batch mode bioreactor with immobilized cells, almost complete ACN removal (>95%) was achieved at a flow rate of 0.1 μl/min ACN and 0.8 μl/min toluene (TOL) (for comparative purposes this is equivalent to 6.9 mg l^?1 h^?1 ACN and 83.52 mg l^?1 h^?1 TOL). In a single-pass mode bioreactor with immobilized cells, at ACN inlet loads of 100–200 mg l^?1 h^?1 and TOL inlet load of ~400 mg l^?1 h^?1, with empty bed retention time (EBRT) of 8 min, ACN removal efficiency was ~90%. The three-dimensional structure and characteristics of the biofilm were investigated using confocal scanning laser microscopy (CSLM). CLSM images revealed a robust and heterogeneous biofilm, with microcolonies interspersed with voids and channels. Analysis of the precise measurement of biofilm characteristics using COMSTAT^® agreed with the assumption that both biomass and biofilm thickness increased along the carbon column depth.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Biochemical basis of the high resistance to oxidative stress in<Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Aerobic organisms experience oxidative stress due to generation of reactive oxygen species during normal aerobic metabolism. In addition, several chemicals also generate reactive oxygen species which induce oxidative stress. Thus oxidative stress constitutes a major threat to organisms living in aerobic environments. Programmed cell death or apoptosis is a physiological mechanism of cell death, that probably evolved with multicellularity, and is indispensable for normal growth and development.Dictyostelium discoideum, an eukaryotic developmental model, shows both unicellular and multicellular forms in its life cycle and exhibits apparent caspase-independent programmed cell death, and also shows high resistance to oxidative stress. An attempt has been made to investigate the biochemical basis for high resistance ofD. discoideum cell death induced by different oxidants. Dose-dependent induction of cell death by exogenous addition of hydrogen peroxide (H₂O₂),in situ generation of H₂O₂ by hydroxylamine, and nitric oxide (NO) generation by sodium nitroprusside treatment inD. discoideum were studied. The AD₅₀ doses (concentration of the oxidants cusing 50% of the cells to die) after 24 h of treatment were found to be 0.45 mM, 4 mM and 1 mM, respectively. Studies on enzymatic antioxidant status ofD. discoideum when subjected to oxidative stress, NO and nutrient stress reveal that superoxide dismutase and catalase were unchanged; a significant induction of glutathione peroxidase was observed. Interestingly, oxidative stress-induced lipid membrane peroxidative damage could not be detected. The results shed light on the biochemical basis for the observed high resistance to oxidative stress inD. discoideum.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

A model for cell type localization in the migrating slug of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> based on differential chemotactic sensitivity to cAMP and differential sensitivity to suppression of chemotaxis by ammonia

The three basic cell types in the migrating slug of Dictyostelium discoideum show differential chemotactic response to cyclic AMP (cAMP) and differential sensitivity to suppression of the chemotaxis by ammonia. The values of these parameters indicate a progressive maturation of chemotactic properties during the transdifferentiation of slug cell types. We present a model that explains the localization of the three cell types within the slug based on these chemotactic differences and on the maturation of their chemotactic properties.     

Bioreactor for solid-state cultivation of <Emphasis Type="Italic">Phlebiopsis gigantea</Emphasis>

Phlebiopsis gigantea fungus used in biological control of root rot is currently cultivated commercially in disposable, sterilizable plastic bags. A novel packed bed bioreactor was designed for cultivating P. gigantea and compared to the plastic bag method and to a tray bioreactor. The spore viability of 5.4 × 10⁶ c.f.u./g obtained with the packed bed bioreactor was of the same order of magnitude as the viabilities obtained with the other cultivation methods. Furthermore, the packed bed bioreactor was less time and space consuming and easier to operate than the tray bioreactor.     

Study of tauopathies by comparing <Emphasis Type="Italic">Drosophila</Emphasis> and human <Emphasis Type="Italic">tau</Emphasis> in <Emphasis Type="Italic">Drosophila</Emphasis>

The microtubule-binding protein tau has been investigated for its contribution to various neurodegenerative disorders. However, the findings from transgenic studies, using the same tau transgene, vary widely among different laboratories. Here, we have investigated the potential mechanisms underlying tauopathies by comparing Drosophila (d-tau) and human (h-tau) tau in a Drosophila model. Overexpression of a single copy of either tau isoform in the retina results in a similar rough eye phenotype. However, co-expression of Par-1 with d-tau leads to lethality, whereas co-expression of Par-1 with h-tau has little effect on the rough eye phenotype. We have found analogous results by comparing larval proteomes. Through genetic screening and proteomic analysis, we have identified some important potential modifiers and tau-associated proteins. These results suggest that the two tau genes differ significantly. This comparison between species-specific isoforms may help to clarify whether the homologous tau genes are conserved. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported by the National Science Foundation of China (30270341; 30630028), the Multidisciplinary Program (Brain and Mind) of the Chinese Academy of Sciences, the Major State Basic Research Program (“973 program”; G2000077800; G2006CB806600; 2006CB911003), the Precedent Project of Important Intersectional Disciplines in the Knowledge Innovation Engineering of the Chinese Academy of Sciences (KJCX1-09-03).     

Influence of medium components on growth kinetics of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Starting with the standard complex medium HL-5C, the influence of different medium components on the growth behavior of Dictyostelium discoideum was investigated. For this purpose, each component was individually deleted from the complex medium HL-5C, and the overall concentration of all components as well as the kinds and concentrations of carbon source were varied. The effects of the supplementation with major components of synthetic medium like vitamins, trace metals, inorganic salts were also investigated. When glucose being the carbon source was deleted from the standard HL-5C medium, the maximal cell density then had a drastic drop. Deletion of yeast extract also caused lower maximal cell density. Of all the carbohydrates investigated only maltose and glucose led to higher final cell concentration. Adding inorganic salts such as CaCl₂ and MgCl₂ to HL-5C medium significantly improved the cell growth, whereas addition of vitamins or trace metals had little effect on cell growth. A semi-empirical model is employed to simulate the cell growth.     

<Emphasis Type="Italic">Funastrum rupicola</Emphasis> (<Emphasis Type="Italic">Apocynaceae:</Emphasis><Emphasis Type="Italic">Asclepiadoideae</Emphasis>), a new species from Bolivia

Summary   Funastrum rupicola Goyder, a new species of Apocynaceae: Asclepiadoideae from Bolivia, is described and illustrated. The conservation status of this species is assessed.     

Degradation of ethylbenzene by free and immobilized <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fluorescens-</Emphasis>CS2

Pseudomonas fluorescens-CS2 metabolized ethylbenzene as the sole source of carbon and energy. The involvement of catechol as the hydroxylated intermediate during the biodegradation of ethylbenzene was established by TLC, HPLC and enzyme analysis. The specific activity of Catechol 2,3-dioxygenase in the cell free extracts of P. fluorescens-CS2 was determined to be 0.428 μmoles min⁻¹ mg⁻¹ protein. An aqueous-organic, Two-Phase Batch Culture System (TPBCS) was developed to overcome inhibition due to higher substrate concentrations. In TPBCS, P. fluorescens-CS2 demonstrated ethylbenzene utilization up to 50 mM without substrate inhibition on inclusion of n-decanol as the second phase. The rate of ethylbenzene metabolism in TPBCS was found enhance by fivefold in comparison with single phase system. Alternatively the alginate, agar and polyacrylamide matrix immobilized P. fluorescens-CS2 cells efficiently degraded ethylebenzene with enhanced efficiency compared to free cell cultures in single and two-phase systems. The cells entrapped in ployacrylamide and alginate were found to be stable and degradation efficient for a period of 42 days where as agar-entrapped P. fluorescens was stable and efficient a period of 36 days. This demonstrates that alginate and polyacrylamide matrices are more promising as compared to agar for cell immobilization.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan