In vitro propagation of <Emphasis Type="Italic">Helleborus</Emphasis> species

A general in vitro cloning system was established for four Helleborus species: H. argutifolius, H. foetidus, H. niger and H. orientalis. The plant material was introduced in vitro from axillary buds. A Murashige and Skoog (MS)—based medium (Murashige and Skoog 1962) was used supplemented with 2% (w/v) sucrose, 2-isopentenyladenine (2-iP) and 6-benzylaminopurine (BA). Multiplication rates depended on the genotype and varied from 1.3 for H. foetidus till 3.8 for H. niger. The first results showed that the rooting phase could be done ex vitro. Rooting was induced by a drench for one week in a solution of indole-3-butyric acid (IBA -3 mg l⁻¹) and 1-naphthaleneacetic acid (NAA-1 mg l⁻¹) at 5°C.     

Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.     

Plant-growth regulators from common starfish (<Emphasis Type="Italic">Asterias amurensis</Emphasis> Lütken) waste

Starfish waste has been shown to be an effective compost material not only in the promotion of plant growth but also in terms of having insecticidal activity. In the present study, plant growth regulation by chemicals from starfish was examined. The aqueous fraction from a hot water extract of the starfish Asterias amurensis Lütken showed plant-growth activity, while the aqueous fraction from a methanol extract inhibited growth of Brassica campestris. The lipophilic fraction from the methanol extract also exhibited a plant growth-promoting effect. The active components from each extract were identified. Asterubine from the hot water extract promoted plant growth. A ceramide from the lipophilic fraction showed root growth promoting effect, and three glucocerebrosides had promotive effects on the entire plant. Asterosaponins were identified as the main growth inhibitors in the aqueous fraction of the methanol extract. These active compounds from starfish waste could be analyzed as potential plant growth regulators in agricultural applications in the future.     

Characterization of calf liver glucosidase I and its inhibition by basic sugar analogs

Glucosidase I, the enzyme catalyzing the first step of N-linked oligosaccharide processing, has been purified from calf liver crude membranes [H. Hettkamp, G. Legler, and E. Bause, (1984) Eur. J. Biochem. 142, 85-90]. Binding experiments with concanavalin A-Sepharose suggest that glucosidase I is a glycoprotein with high-mannose carbohydrate chain(s). The enzyme has a subunit molecular mass of approximately 83 kDa and specifically hydrolyzes the terminal alpha-1,2-linked glucose residue from the natural Glc3-Man9-GlcNAc2 oligosaccharide. Studies with a variety of substrates modified in the aglycon moiety suggest that the Glc2 branch rather than the more distant domains of the substrate molecule are important for binding and hydrolysis. Glucosidase I does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (dNM) and its N-alkyl derivatives. Ki values range from 0.07 microM for N-methyl-dNM to 1.0 microM for dNM, measured at the pH-optimum of enzyme activity. The pH dependence of inhibition indicates that the cationic form of the inhibitors is the active species. Comparison of the Ki for N-decanoyl-dNM (approximately 70 microM) with that of N-decyl-dNM (approximately 0.4 microM) suggests that electrostatic interactions at the catalytic site of the enzyme are important for inhibitor binding. 1-Deoxymannojirimycin, previously assumed to be a specific mannosidase inhibitor, as well as its N-methyl and N-5-carboxypentyl derivatives, inhibit glucosidase I with Ki values around 190, 17, and 100 microM, respectively. This apparent lack of specificity shows that in vivo experiments on N-glycoprotein processing as well as the interpretation of results with these mannosidase inhibitors may give misleading results when these compounds are used in the millimolar range.     

UDP-Gal: <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

As compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis (VL) has received limited attention. In this study, we demonstrate for the first time that an UDP-Galactose: N-acetylglucosamine β 1–4 galactosyltransferase (GenBank Accession No. EF159943) expressing attenuated LD clonal population (A-LD) is able to confer protection against the experimental challenge with the virulent LD AG83 parasite. A-LD was also effective in established leishmania infection. The vaccinated animals showed both cell mediated (in vitro T-cell proliferation, and DTH response) and humoral responses (Th1 type). These results demonstrate the potential of the attenuated clones as an immunotherapeutic and immunoprophylactic agent against visceral leishmaniasis.